Effect Of Four Traditional Chinese Medicines On The Expression Of Cyclooxygenase Gene And EP4 Receptor Genes In Murine Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) have the function of reducing inflammation, controlling pain and anti-carcinogenesing by inhibiting the activity of cyclooxygenase (COX) which is the the rate-limiting enzyme producing prostaglandin in mammalian. In this thesis, four Traditional Chinese Medicine (TCM) extracts including ginsenoside Rb1, Re and Rg1 and cryptotanshitone have been chosen to study their effects on the expressions of COX gene and prostaglandin E2 (PGE2) receptor EP4 gene in murine RAW264.7 cell line, and the results obtained from these experiments are as follows. Firstly, the total RNA was extracted from the murine RAW 264.7 cells being treated with lipopolysaccharide (LPS) for 6 hours to induce the COX gene expression, then ginsenoside Rb1, Re, Rg1 and cryptotanshitone added to the medium to stimulate cells for another 18 hours, respectively. The qualitative RT-PCR technique was applied for the extracted total RNA as the templates and the bands of the COX-2 cDNA fragment as well as a putative novel COX family gene called COX-3 were obtained. The results showed that ginsenoside Rb1 inhibits the expression of COX-3 specifically, while ginsenoside Re, Rg1 and cryptotanshitone have no effect on the expression of COX-3. From our experimental results, four drugs are all have no effect on COX-2 gene expression.Secondly, a quantitative RT-PCR was performed to obtain the bands of EP4 receptor and β-actin genes' fragments together by agarose gel analysis. The activities of EP4 receptor were decreased from the total value to 62% and 39% with stimulating by ginsenoside Rb1 and Re, respectively. Furthermore, crptotanshitone and ginsenoside Rg1 have been found to inhibit the expression of EP4 receptor gene completely. A quantitative PCR was used to amplify the expected DNA sequences from total RNA template obtained from murine RAW 264.7 cells induced for COX-2 expression, and then treated with ginsenoside Rb1. The activities of EP4 receptor gene expression were increased during the beginning hour decreased from the second hour and until twenty-four hours later.Finally, our experimental results show that ginsenoside Rb1 inhibit the expression of a novel cyclooxygenase and its effect mechanism is different from the COX-2 specific inhibitors. Thus ginsenoside Rb1 is becoming the one of stimulators which have the potential of exploring new kind of NSAIDs.