Low-temperature Plasma Promotes Fibroblast Proliferation in Wound Healing by ROS-activated NF-κB Signaling Pathway

Low-temperature plasma(LTP)has shown great promise in wound healing,although the underlying mechanism remains poorly understood.In the present study,an argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro and skin wounds in BALB/c mice.The in vitro analysis revealed that treatment of fibroblasts with LTP for 15 s resulted in a significant increase in cell proliferation,secretion of epidermal growth factor(EGF)and transforming growth factor-β1(TGF-β1),production of intracellular reactive oxygen species(ROS),and the percentage of cells in S phase,protein expression of phosphorylated p65(P-p65)and cyclin D1,but a noted decrease in the protein expression of inhibitor kappa B(IκB).The in vivo experiments demonstrated that 30-s LTP treatment enhanced the number of fibroblasts and the ability of collagen synthesis,while 50-s treatment led to the opposite outcomes.These results suggested that LTP treatment promotes the fibroblast proliferation in wound healing by inducing the generation of ROS,upregulating the expression of P-p65,downregulating the expression of IκB,and activating the NF-κB signaling pathway and consequently altering cell cycle progression(increased DNA synthesis in S phage).

Fibroblast proliferation alters cardiac excitation conduction and contraction： a computational study

In this study, the effects of cardiac fibroblast proliferation on cardiac electric excitation conduction and mechanical contraction were investigated using a proposed integrated myocardial-fibroblastic electromechanical model. At the cellular level, models of the human ventricular myocyte and fibroblast were modified to incorporate a model of cardiac mechanical contraction and cooperativity mechanisms. Cellular electromechanical coupling was realized with a calcium buffer. At the tissue level, electrical excitation conduction was coupled to an elastic mechanics model in which the finite difference method （FDM） was used to solve electrical excitation equations, and the finite element method （FEM） was used to solve mechanics equations. The electromechanical properties of the proposed integrated model were investigated in one or two dimensions under normal and ischemic pathological conditions. Fibroblast proliferation slowed wave propagation, induced a conduction block, decreased strains in the fibroblast proliferous tissue, and increased dispersions in depolarization, repolarization, and action potential duration （APD）. It also distorted the wave-front, leading to the initiation and maintenance of re-entry, and resulted in a sustained contrac- tion in the proliferous areas. This study demonstrated the important role that fibroblast proliferation plays in modulating cardiac electromechanical behaviour and which should be considered in planning future heart-modeling studies.

Experimental study on effect of endothelin in the proliferation and collagen synthesis of human scar-derived fibroblasts

Objective To investigate the role of endothelin(ET) in the proliferation and collagen synthesis of human scar-derived fibroblasts and the moduktion of its antagonists such as nitric oxide(NO), tetrandrine ( Tet). Methods With the cultured fibroblasts from the scarring tissue, the cell pdiferation was determined by[3H]-TdR incorporation, while the collagen synthesis was evaluated by[3H]-proline incorporation. Results The ET-1 was significantly increasing the proliferation and collagen synthesis of human scar-derived fibroblasts. The values of [3H]-TdR absorption in the 2.5 ng/ml,25 ng/ml and 100 ng/ml of ET-1 groups were 1.8 times,4 times and 4.9 times more than in the control group, respectively(P【0. 01),while the values of the [3H]-proline incorporation were 1.1 times,3.1 times and 3.8 times respectively(P【0.01). The fibroblasts, treated with 50 μg/ml of S-nitroso-N-acetyl penicillamine(SNAP), were no detectable effect on the basal level of DNA synthesis,but produced decreasing effect on the

EFFECTS OF CALCIUM AND CALMODULIN INHIBITORS ON THE ABNORMAL PROLIFERATION OF LUNG FIBROBLAST

The effects of alveolar macrophage (Am) conditioned media from interstitial lung disease (ILD) patients on fibroblast (FB), and the role of calcium (Ca2+) blockers and calmodulin (CaM) inhibitors on the proliferation of lung FB were studied. We found that the AM conditioned media could stimulate FB cell proliferation and this effect could be abolished by Ca2+ blockers and CaM inhibitors. The results indicated that AM was in activated state in ILD and released some kinds of cytokines to stimulate the proliferation of FB, and Ca,2+ CaM were partially responsible for these actions.

The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor

Objective To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).Methods We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using 32P-labelled substrate. In the primary culture of lung fibroblasts, 3H-thymidine (3H-TdR) and 3H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DMA and collagen synthesis stimulated by bFGF.Results We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1±2.0 pmol Pi/mg pr/min). CsA (10^(-8)-10^(-6) mol/L) inhibited lung fibroblast 3H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20% , 46% and 66% (P< 0.01). CsA (10^(-7) -10^(-6) mol/L) inhibited 3H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P<0. 01). In a culture medium, CsA (10^(-8)-10^(-6) mol/L) inhibited 3H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P<0. 05) and 56% (P<0. 01). CsA (10^(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P<0. 01).Conclusions Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

Effects of Tetramethylpyrazine and Radix Salviae Miltiorrhizae on Collagen Synthesis and Proliferation of Cardiac Fibroblasts

Objective: To explore the effects of Tetramethylpyrazine (TMP) and Radix Salviae Miltiorrhizae (RSM) on collagen synthesis and proliferation of cardiac fibroblasts. Methods: Using collagenase and pancreatin digested rat cardiac tissue assay to isolate cardiac fibroblasts (FB). Different dosage of TMP, RSM and norepinephrine were used to study their effects on the collagen synthesis and proliferation of cultured cardiac FB. Results: Compared with the control group, moderate or high dosage TMP and RSM could significantly inhibit the collagen synthesis and the proliferation of cultured cardiac FB. Moreover, low-dose TMP (50 mg/L) and low-dose RSM (3 g/L) could antagonize the collagen synthesis and the proliferation of cultured cardiac FB stimulated by NE (500μg/L). Conclusion: Both TMP and RSM can inhibit the collagen synthesis and proliferation of cultured cardiac FB processes.The mechanisms of these effects might be correlated to their Ca++ antagonistic action. Original article on CJIM(Chin) 1998; 18(7): 423

Transforming growth factor-β1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit the activity of keloid fibroblasts

Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.

Differentiation of NIH3T3 fibroblasts into adipocytes induced by peroxisome proliferator activated receptor γ2 expression

Objective To express mouse peroxisome proliferator activated receptor γ2 (mPPARγ2) in NIH3T3 fibroblasts mediated by the recombinant retrovirus and study its function.Methods The mPPARγ2 gene was subcloned into retrovirus vector pGCEN to generate the recombinant pGCEN/mPPARγ2. Then it was packaged into PA317 cells and selected with G418. Viral supernatants were harvested and then used to infect NIH3T3 fibroblasts. PPARγ activator 5,8,11,14-eicosatetraynoic acid (ETYA) was used to induce the mPPARγ2-expressing NIH3T3 cells into adipocyte differentiation.Results The recombinant retrovirus pGCEN/mPPARγ2 was constructed, and the higher titers of the viral supernatants were obtained. mPPARγ2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus. Lipid accumulation obviously existed in these induced adipocytes which morphologically resembled mature adipocytes in vivo and expressed tissue specific adipocyte P2 (AP2) and Leptin genes.Conclusions An adipocyte differentiation model in vitro was successfully established. The work is the basis for further research on the molecular mechanism of adipocyte differentiation induced by PPARγ2.