Study On The Mechanism Of Signaling Pathway Of MMP-2and CTGF Expression Induced By Arecoline Effecting On Oral Mucosal Epithelial Cell And Fibroblast

PART I:Effects of arecoline on proliferation, apoptosis and DNA damage of primary oral mucosal epithelial cell and fibroblastObjective:This study was conducted to indicate the effect of arecoline on proliferation, apoptosis and DNA damage of primary culturing oral mucosal epithelial cells and fibroblastMethods:Oral mucosal epithelial cell and fibroblast were cultured and amplified in vitro, and identified by cell morphology and keratin and vimentin staining. MTT assay was applied to detect the effect on proliferation of epithelial cell and fibroblast by high concentration arecoline intervening in short time and low concentration arecoline intervening in long time. Flow cytometry was applied to detect the effect on cell cycle of epithelial cell and fibroblast by arecoline intervening. Comet assay was applied to detect the effect on DNA damage of epithelial cell and fibroblast by arecoline intervening.Results:The cultured cells had typical morphology of epithelial cell and fibroblast, and keratin and vimentin staining was positive. After intervened by high concentration (0.2mM-0.8mM) arecoline in short-term (0-72h), the activity of epithelial cell decreased and morphological change with increasing time and concentration of arecoline and the difference was statistically significant (P<0.05), the activity of fibroblast decreased and morphological change was no obviously and the difference is not statistical significance (P>0.05).0.8mM arecoline intervened epithelial cell for48hours resulted in arresting in the G2/M phase compared with the control group and the difference was statistically significant (P<0.05), and the effect of0.8mM arecoline on fibroblast cycle was not obvious and the difference is not statistically significant (P>0.05). After intervened by0.6mM arecoline for24hours,the apoptosis of epithelial cell with a dose and time dependent and the difference was statistically significant (P<0.05), but the apoptosis of fibroblasts is not obvious and the difference is not statistically significant (P>0.05).Low concentration(0.1mM)arecoline significantly inhibited epithelial cell growth for12days and the difference was statistically significant (P<0.05), the same concentration arecoline with same time intervention on fibroblast growth had no obvious effect and the difference was not statistically significant (P>0.05). Different concentration arecoline intervened fibroblast for24hours, the DNA damage of fibroblast was not obvious comparing with the control group(P>0.05),and DNA damage of epithelial cell was obvious with concentration dependent,comparing with the control group and the difference was statistically significant (P<0.05), and the higher the concentration, the more serious damage of DNA.Conclusion:1. The high concentration arecoline intervention in short time could inhibit the proliferation of epithelial cell, induced apoptosis and arresting in G2/M phase of cell cycle, and the DNA damage showed a dose-response dependence.2. The high concentration arecoline intervening on fibroblast in short time had no obvious effect on proliferation, apoptosis and DNA damage.3. After12days intervened by low concentration arecoline,the number of viable cell of epithelial cell was significantly decreased, whereas number of viable cell of fibroblast had no obvious change. PART Ⅱ Effect of arecoline on expression of MMP-2in epithelial cells and its mechanism of signaling pathwayObjective:To investigate the effect of arecoline on expression of MMP-2in epithelial cell MMP-2and its possible mechanism.Methods:Western blotting, RT-PCR and gelatin zymography were applied to detect the expression of MMP-2in epithelial cell after intervened by high concentration arecoline (0.2mM-0.8mM) in short time (10minutes) and a low concentration arecoline (0.1mM) in long time(24hours).ERK, PI3K, p38MAPK, c-JNK and NF-κ beta signaling pathway inhibitors,namely PD98059, LY294002, U0126, N-acetyl-L-cysteine (N-acetyl-L-cysteine, NAC), SB203580, SP600125and Bayl1-7082were pretreated epithelial cell before it was intervened by0.0mM arecoline so as to reveal the mechanism of signal pathway of effect on MMP-2expression by arecolineResults:MMP-2expression in epithelial cell was significantly increased with concentration dependent after intervened by high concentration arecoline (0.2mM-0.8mM) in short time (10minutes) and cultured for12hours, and the difference was statistically significant (P<0.05) comparing with the control group. The expression of MMP-2was significantly higher than that of the control group and the difference was statistically significant (P<0.05) after intervened by low concentration arecoline (0.1mM) in long time (24hours) and cultured for24hours. PI3K signal pathway inhibitor, LY294002could inhibit the ability of MMP-2expression induced by low concentration (0.1mM) arecoline with a dose-dependent manner.ERK signal pathway inhibitor, PD98059had no effect. P38MAPK, c-JNK and NF-κ beta signaling pathway inhibitors,SB203580, SP600125, Bay11-7082could inhibit the ability of MMP-2expression induced by low concentration (0.1mM) arecoline, while NAC and U0126had no such effectConclusion:1. The high concentration and low concentration arecoline in long time or short time intervening epithelial cell could lead to up regulate the expression of MMP-2protein.2. The up-regulating mechanism of MMP-2of epithelial cell intervened by low concentration arecoline in long time may be related to PI3K, MMP-2protein p38MAPK, c-JNK and NF-beta signal pathway. PART III Effect of arecoline on expression of CTGF in fibroblast and its mechanism of signaling pathway and the reversal effect of curcuminObjective:To observe CTGF expression of fibroblast intervened by arecoline and explore the mechanism of curcumin effecting on CTGF expression of fibroblast induced by of arecoline.Methods:Immunohistochemical method was applied to detect the CTGF expression in20cases of oral submucous fibrosis. Western blot was applied to detect the effect on expression of CTGF induced by arecoline in long time and short time,and signal pathway inhibitors of PD98059(12.5,25,50μM), SB203580(10μM), SP600125(10μM), Bay11-7082(10μM) and NAC (5mM) were applied to observe the mechanism of signaling pathway on CTGF expression induced by arecoline. Curcumin was applied to detect its reversal effect on expression of CTGF induced by arecoline.Results:The expression of CTGF in patients with oral submucous fibrosis was significantly higher than that in normal mucosa,and the difference was statistically significant (P<0.05). After intervened by different concentration arecoline, western blot assay showed that arecoline promoting the expression of CTGF. After intervened by0.1mM arecoline for2hours, CTGF protein increased about2.5times,and it was the highest,then decreased. Western blot was applied to detect the expression of CTGF intervened by different signaling pathway inhibitors, Bayl1-7082, SP600125, SB203580and NAC significantly decreased the expression of CTGF induced by arecoline, while PD98059had no effect. Western blotting showed that after curcumin early intervention,fibroblast decreased expression of CTGF induced by arecoline, and curcumin decreased CTGF expression with dose-dependent manner.Up to concentration, the expression of CTGF was lower than that in the control groupConclusion:1. Expression of CTGF in oral submucous fibrosis was significantly higher than that in control group.2. Fibroblast increased expression of CTGF induced by arecoline was in effect and time dependent manner, and related with NF-kβ,JNK, p38MAPK signal pathway.3. Curcumin could reverse the CTGF expression of fibroblast induced by arecoline.