Mechanistic Study Of Anti-inflammatory Effect Of Bicyclol And Exploring The Interaction Between Bicyclol And Inosine 5’-monophosphate Dehydrogenase Ⅱ

The aim was to investigate the potential target and the anti-inflammatory mechanisms of bicyclol, which has been extensively used in clinic for decades in China. Virtual molecular system TarFisDock showed that inosine 5’-monophosphate dehydrogenase II(IMPDH II) has the highest probability of binding to bicyclol. To investigate the possible role of IMPDH II played on the mechanisms of bicyclol, mice splenic lymphocyte, human lymphocyte and recombinant enzyme models were used. Bicyclol (1 μM and 5 μM) significantly inhibited the proliferation of mice splenic lymphocytes stimulated by concanavalin A (conA). However, bicyclol did not show significant inhibitory effects on proliferation of human peripheral blood mononuclear cells (hPBMC) induced by phytohemagglutinin (PHA) (only two experiments). IMPDH Ⅱ enzyme kinetic model showed that bicyclol only had a slight regulatory effect on IMPDH Ⅱ enzyme activity. These results revealed that bicyclol not like mycophenolic acid, maybe not a conventional inhibitor of IMPDH Ⅱ. Further studies showed that bicyclol affected the biological function of IMPDH Ⅱ. Bicyclol could promote the active toll-like 2 receptor (TLR2) complex to recruit IMPDH Ⅱ, then reduce nuclear factor κB (NF-κB) expression, increase I-κB expression and decrease cytokine release, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β). It may be a new mechanism of bicyclol for its anti-inflammatory effect. But the detail mechanism of bicyclol is still obscue. The results of part 1 showed that bicyclol could promote TLR2 recruiting IMPDHⅡ to inhibit the activation of NF-κB pathway downstream. Latest clinical trials showed us that bicyclol also has curative effect on the Non-alcoholic Fatty Liver Disease (NAFLD). In this part, we furtherly investigate the inflammatory effect and the effect of bicyclol on lipid-activated nuclear receptors in pam3-stimulated THP-1 derived macrophages. Firstly, the proper concentration of PMA which could stimulate THP-1 monocytes differentiating into macrophages was studied.50ng/mL PMA treating cells for 2 days was applied for differentiation.5,10,50μM bicyclol could decrease cytokines TNF-a, IL-6, IL-1β levels induced by pam3. The results of western blot showed that bicyclol could decrease the expression of PI3K, depress the phosphorylation of Akt, IKK, IκB and prevent the nuclear entry of NF-κB. Bicyclol also could increase the expression of PPARy in dose dependent manner.As the ligand of toll-like 4 receptor, LPS was used to stimulate the macrophage. The results showed that bicyclol could significantly inhibit the pro-inflammatory cytokines (TNF-a, IL-1β, IL-6) in LPS-stimulate macrophages. It could decrease the expression of PI3K, the degradation and phosphorylation of IκB, the nuclear entry of NF-κB. But it had no significant effect on the expression and phosphorylation of Akt and IKK. Then, we treated macrophages with rosiglitazone and bicyclol respectively, without stimulation of LPS or pam3. The results showed that bicyclol could up-regulate the expression of PPARy and IκB significantly. 10μM bicyclol shows the equivalent effect comparing to 10μM rosiglitazone. PPARγ can bind to initiator of IκB gene to promote transcription of IκB. After using siRNA to knockdown the expression of PPARγ, we found that bicyclol could still up-regulate the expression of PPARγ and IκB. But the activation effect was weakened. It reminded us that the activation of bicyclol on IκB expression might due to the promotion of bicyclol on PPARγ expression. And inhibition effect of bicyclol on proinflammatory cytokines release were weakened as well, including TNF-α, IL-1β. It reminded us that the activation of PPARγ just might partly contribute to the anti-inflammatory effect of bicyclol.