| [Background] Cyclooxygenase(COXs) is the key rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids. There are at least two isoforms of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed in almost all mammalian tissues and cells, promoting the PG synthesis and playing important roles in maintaining and regulating the physiological function of the platelet, endothelial cell, kidney and stomach. However, COX-2, rarely physiologically expressed in normal tissues, can be induced by the inflammatory factors or cytokines such as IL-1, TNF, TGF-a, IFN-γ,PAF and endotoxin and some carcinogens. It is demonstrated by some researches that the expression of COX-2 is increased from normal liver tissue, chronic hepatitis to cirrhosis, while the strongest COX-2 expression is in the cirrhosis tissues not in the carcinoma tissues. Some other investigations also reveal that COX-2 is upregulated in the cirrhosis tissues adjacent to the cancers and in the highly-differentiated cancers as well. In the experimental liver cirrhosis models induced by choline-deficient, L-amino acid-defined diet (CDAA) and thioacetamide(TAA), the expression of COX-2 is in ascent. The inhibitor of COX-2, effectively inhibiting the COX-2 expression, can introvert the fibrosis and entirely block the development of liver cirrhosis. COX-2 is expressed at high level in the different stage of liver fibrosis and in chronic alcoholic hepatitis; COX-2 is also upregulated in Kuffer cells, liver cells and hepatic stellate cells(HSC). As activated hepatic stellate cells play a crucial role in the pathogenesis of liver inflammation and fibrosis, There is a evidence shows that cultivated HSC expresses COX-2 protein in vitro and a COX-2 selective inhibitor can inhibit a-smooth muscle actin protein expression in a dose-dependent manner. These indicate that COX-2 may participate in the liver fibrosis and it may be a potential target for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC. But the precise role of COX-2 in hepatopathology and how COX-2 inhibitors regulate the activity and proliferation of HSC or the mechanism involved remains unclear.[Objectives] By using the non-specific inhibitor of COX-2— Indomethacin and the specific inhibitor—Celecoxib, we investigate their effects on the proliferation and activation of the fibroblast, the cell cycle and apoptosis and ECM. We also interested in the expressions of the COX-2, FN and LN after the cells were treated with these inhibitors. Thus to explore the antifibrotic function of the COX-2 inhibitors.[Methods]We substitute the NIH-3T3 cell for the HSC. (1)Western blot was used to detect the expressions of COX-2, FN and LN. (2) Examine the effect of the COX-2 inhibitors on the proliferation and activation of the NIH/3T3 cells by using MTT assay. (3) Observe the cell toxic and morphologic effectof the COX-2 inhibitors under the reverse microscope. (4) FACS was applied to detect their effect on the cell cycle. (5) Apoptosis of the cells was determined by Annexin V-FITC staining.(6) Immunoradioactivity assay was used to detect the effect of the inhibitors on the ECM. [ Results ] 1. COX-2 was positively expressed in the NIH-3T3 cells; 2. The cell growth curve by MTT assay showed that Indomethacin (5-10μmol/L) and Celecoxib (50-200μmol/L) can, without evident cell toxic effect, inhibit the cell growth (p<0.001), in a dose and time-dependent way, While the inhibition was inapparent at the first 24h after administration. The IC50 of each was 189.56μmol/L and 36.78μmol/L. 3. FACS revealed that cell fraction of G0/G1 and S phase was reduced from 0.677 to 0.163, 0.268 to 0.103, respectively, while cell fraction of G2/M increased from 0.05 to 0.734 after treating with Celecoxib(40μmol/L) for 72-120hrs. However, after the cells were administered with Indomethacin(200μmol/L) for 2h-120h, the cell fraction of G1 and S phase was increased from 0.677 to 0.77 and reduced from 0.268 to 0.161. Cells were blocked at Gl phase. 4. Annexin V-FITC staining showed that cells were subjected to apoptosis after being treated with Indomethacin and Celecoxib, with the apoptosis rates 18.4% and 22.3%, compared to the negative control 2%. 5. Immunoradioactivity assay revealed that HA and LN in the supernatant of NIH3T3 were reduced gradually as the increasing administration of Celecoxib. The inhibition rates of the LN by Celecoxib(5-40nmol/L) were 19.73%, 18.15%, 22.05% and 24.64% and the inhibition rates of the HA by Celecoxib(20-40μmol/L) were 17.53% and 24.24%. Both of these inhibitions functioned in a dose-dependent way and...
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