Rhizoma Coptidis Inhibits LPS-induced MCP-1 Production In Murine Macrophages

Objective1. To study the effect of the extract of Rhizoma Coptidis on LPS-induced inflammatory activity in a murine macrophage cell line and its molecular mechanism.2. To explore the main compound in the extract of Rhizoma Coptidis by which it executes its anti-inflammatory function and to compare the effect with other common anti-inflammatory drugs.Methods1. Treated LPS stressed RAW 264.7 with different concentration of the extract of Rhizoma Coptidis and incubate at different time points then detect the activity of transcription factor(AP-1 and NFκB) in the nuclear extracts with the help of EMSA , in the meanwhile the MCP-1 concentration in the supernatant is tested by ELISA.2. Treated LPS stressed RAW 264.7 with different anti-inflammatory drugs(Statins, ACEI and ARB) and incubate at different time points then detect the activity of transcription factor(AP-1 and NF ?B ) in the nuclear extracts with the help of EMSA , in the meanwhile the MCP-1 concentration in the supernatant is also tested by ELISA. Results1. Morphology of RAW 264.7 cells did not differ between control cells and cells, preincubated with various dilutions of Rhizoma coptidis extract.2. Incubation of LPS-stimulated RAW cells with Rhizoma coptidis inhibited AP-1 and NFκB activity in a concentration and time-dependent fashion. Significant reduction of transcription factor NFκB activity required higher concentrations and longer preincubation times with Rhizoma coptidis extract, as compared with effects on AP-1 activity.3. Incubation with the main alkaloid compound of Rhizoma coptidis, berberine, also significantly inhibited binding activity of AP-1 and NFκB.4. HMG-Co enzyme A reductase inhibitors (statins) as well as ACE-inhibitors and angiotensin receptor blockers (ARBs) are known for their potent anti-inflammatory effects. Preincubation with the ARB olmesartan resulted in a small, but significant reduction of AP-1 activity in LPS-stimulated RAW cells however, the effect was by far less profound as compared with Rhizoma coptidis extract and with berberine. All other tested statins and ARBs failed to show significant effects on AP-1 activity. No significant effects on NFκB activity were observed with any of the tested statins and ARBs,5. Secretion of monocyte chemoattractant protein-1 by LPS-stimulated RAW 264.7 cells was also significantly reduced by preincubation with Rhizoma coptidis and berberine in a concentration- and time dependent fashion,as evaluated by ELISA of supernatants. Again, preincubation of stimulated cells with various statins and ARBs did not inhibit LPS-induced secretion of MCP-1 by RAW cells. In contrast to the results of the transcription factor essays, MCP-1 secretion was significantly inhibited already after pre-treatment with low concentrations of Rhizoma coptidis or berberine. Conclusions1. The extract of Rhizoma Coptidis can significantly inhibit the inflammatory activity in LPS induced RAW 264.7 cells.2. Berberine represents the main anti-inflammatory compound of Rhizoma coptidis,and its anti-inflammatory effect is much more stronger than the Statins ,ACEIs and ARBs.3. The anti-inflammatory effect of Rhizoma coptidis or berberine is at least partially mediated by the inhibition of MCP-1 secretion The inhibitory effects on cytokine secretion(MCP-1) might not exclusively be mediated via AP-1 and NFκB.