Study Of PI3K/Akt Signal Pathway In The Human Hypertrophic Scar Fibroblast In Vitro And Its Correlation With Connective Tissue Growth Factor

Hypertrophic scar(HS), characterized by excessive fibroblast(Fb) proliferation and local collagen deposition, is a very common pathologic phenomenon of scar hyperplasia after human skin injury and wound healing, and it's pathogenesis is still unclear. The Fb, extracellular matrix and the cytokine are very important during the process of HS. Now how to inhibit the proliferation and induce the apoptosis of Fb under the regulation of cytokine is the main problem in HS research area.Connective tissue growth factor(CTGF) is a kind of important cytokine during the process of tissue fibrosis. The over expression of CTGF is very closely related with some proliferative and fibrotic diseases, such as scleroderma,renal fibrosis,cirrhosis of liver,pulmonary fibrosis and atherosclerosis, etc. Formerly, our experiments demonstrate that the expression of CTGF protein and mRNA in the HS tissue is higher than that of in normal skin and flat scar. CTGF can promote the proliferation and transdifferentation and avoid apoptosis of HS Fb. However, its signal pathway of promoting fibrosis is still unclear.PI3K/Akt signal pathway plays an important role in the maintenance of the normal physiological functions such as growth, differentiation, metabolism and rearrangement of the cytoskeleton so on. Phosphatidylinositol 3 - kinase (PI3K) is an intracellular signal transduction molecule as the second messenger. Akt , also known as protein kinase B (PKB), is a serine / threonine protein kinase, and is the downstreamer of PI3K during the signal chain. Akt can deliver the signal from PI3K into the target molecule such as PCNA, NF-κB etc in cell nuclear, mainly by the serine / threonine residues of the substrate phosphorylation .So far, the relationship between the PI3K/Akt signal transduction pathway and HS fibrosis induced by CTGF is still unclear. Liu Yuli reported that the expression of phosphorylized -Akt (p-Akt)in the HS tissue was higher than that of in the flat scar and normal skin tissue by the immunohistochemical staining detection.For the further exploration of the relationship between PI3K/Akt signal pathway and CTGF in HS fibrosis, we observe if CTGF can activate the PI3K/Akt signaling pathway, and which biological effects are mediated by PI3K/Akt signal pathway. The results will be helpful to explain the effect of CTGF on the pathogenesis of the HS.The major experiment contents and conclusions were as follows:1. Firstly HS Fb and normal skin Fb were cultured in vitro, then PI3K proteins and mRNA expression were detected with the methods of immunocytochemistry staining and reverse transcription polymerase chain reaction (RT-PCR). Compared with normal skin Fb, there was over expression of PI3K in HS Fb.2. Secondly we stimulated cultured HS Fb with 10 ng/ml exogenous recombinant human CTGF in order to detect the expression of signal molecule p-Akt by Western Blot method in the different time (0 min, 5min, 15min, 30min, 60min, 120min) . In 15 min, the level of phosphorylized Akt was higher than that of the control group. Akt phosphorylation reached the peak in 30 min, and sustained over at least 120 min. Next, we stimulated HS Fb with different concentration (2.5 ng / ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml) exogenous recombinant human CTGF in order to observe the dose-dependent effect in 30min. With the increase of CTGF concentration, the activation of Akt phosphorylation levels also increased. 20ng/ml CTGF owned the greatest activation on Akt phosphorylation. Then, different concentration (1μmol / L, 10μmol / L, 20μmol / L, 50μmol / L) PI3K's specific inhibitor LY294002 was used to block the PI3K/Akt signal pathway induced by CTGF. The results showed that with the increase of LY294002 concentration, the phosphorylation level of Akt declined gradually. 50μmol / L concentration of LY294002 completely blocked the activation of the PI3K/Akt signal pathway induced by CTGF. From this part, we can conclude that CTGF can activate the PI3K/Akt signal pathway specificly.3. Finally, we observed the biological effect of PI3K/Akt signal pathway duing the HS fibrosis induced by CTGF. HS Fb cultured in vitro was treated with 50μmol / L LY294002 in advance, then stimulated with 20ng/ml CTGF. MTT method was used to detect the proliferation of HS Fb. Western Blot method was used to detect the expression of a- smooth muscle actin which is a symbol of myo- Fb. Flow cytometry PI and Annexin V staining method was used to detect HS Fb apoptosis. Immunocytochemistry staining method was used to detect the expression of PCNA. Western Blot method was used to detect the expression of NF-κB. The results were the following: LY294002 only blocked part of HS Fb proliferation and transdifferentiation which were caused by CTGF. LY294002 promoted HS Fb apoptosis which was blocked by CTGF. At the same time, compared with the control group, the expression of PCNA and NF-κB increased significantly after CTGF stimulation.So we concluded that there is high level expression of PI3K in the HS Fb in vitro. CTGF can activate specificly PI3K/Akt signal pathway with the dose-dependent effect. 50μmol / L LY294002 can completely block the activation of PI3K / Akt signal pathway induced by 20ng/ml CTGF. PI3K / Akt signal pathway mediates the proliferation, trasdifferentiation, and apoptosis of HS Fb induced by CTGF. These effects may be caused by increasing the expression of PCNA and NF-κB. There may be other signal pathways which mediate the HS Fb proliferation and trasdifferentiation induced by CTGF.