Synthesis And Its Immunoloregulation Mechanism Research Of Pseudolaric Acids B

Objective With the changing of environment and climate,as well as social and economic development,the autoimmune diseases caused by immune system overreacts are increasing quickly,they seriously harm to human health and affect the quality of life.Commonly used immunosuppressive drugs include corticosteroids,calcineurin,antihistaminic agents and so on.But the high level toxicity and side effect and expensive price limit the clinical application of the drugs.The Pseudolaric acids B refining from Pseudolarix amabilis which is a kind of classical Chinese medicine has good immunomodulatory activity,but its mechanism of action is not totally clear so far and it still has high level toxicity and side effect in case of long term using.So that in this research,we make structural modification of PB to obtain derivatives,and then evaluate the inhibition of lymphocyte proliferation activity,finally explore the therapeutic effect of PB used on delayed-type hypersensitivity(DTH)model mice and its possible mechanism of action,in order to get new drugs with high efficiency,low toxicity and lay the foundation for the development of new immunosuppressive agents.Methods 1.PB reacted with aliphatic alcohol or amine with N,N-dialkyl tertiary to get the new compouds of C-18 position reformed.2.Deacetyl derivative of PB reacted with aliphatic alcohol or amine with N,N-dialkyl tertiary to get the new compouds of C-18 position reformed.3.Indole derivatives were designed and synthesized to find anti-proliferation activity.4.The proliferation activity of the derivatives of PB to T/B lymphocytes cells and its cytotoxic activity towards nomal lymphocytes were tested in vitro by MTT assay.Then lymphocyte toxicity was analysized and compared mofetil.5.The pharmacodynamic of PB were discussed in DNFB-induced DTH mouse.6.Using the RT-PCR and Western Blot methods to detect PB regulate DTH mice related factors and expression of signaling pathways.Results Fifteen PB derivates and four indole derivates were synthesized.Among them,10 compounds were got by modified C-18 position,five compounds were synthesized by C-4 deacetyl derivative of PB reacting with aliphatic alcohol or amine with N,N-dialkyl tertiary.Four compounds were got by modified of indole.All the structures were confirmed by 1H-NMR,and HR-ESI-MS.The activity evaluation result showed that,all the compounds show a low cytotoxic activity,some compounds have higher immunosuppressive activity to T lymphocytes cells than mycophenolate mofetil(MMF).After the hydrolysized of C-4 acetyl,anti-proliferation activity was no better than before.PB could improve the body weight decreasing problem of DTH mice which caused by DNFB.Moreover,PB decreased the thymus index and spleen index,thus suppressed the immune organ enlargement.PB also decreased ear swelling,improved the ear tissue inflammation.PB could decrease the gene expression of IL-22,IL-22R and inhibit the immune response,and increase the expression of IL-10,IL-10R,thus promoting its immunomodulatory effects,inhibiting immune and inflammation response,relieve hyperactive immune state.PB could also decrease the expression of IL-β,TNF-α,inhibit the inflammatory response disorders exacerbation and decrease the expression of heat shock protein HSP27,thus regulating their adaptation to external stimulation,as a result the body could adapt to outside interference rapidly,and play an active defensive role toward enviromental interference and stimulation.Furthermore,PB could inhibit the phosphorylated activation of MAPKAPK-2,and increase the expression of PPARγ,resulting in the synergistic effects in the regulation of immune and inflammatory net.Conclusion PB derivatives with a structure of N,N-dialkylamino aliphatic amine or alcohol have good anti-proliferation activity to T lymphocytes.After hydrolysized the C-4 acetyl,the anti-proliferation activity is lower than before,so that C-4 acetyl is necessary for anti-proliferation activity.PB has a better therapeutic effect to DTH mice.Our further study will focus on the synthesis of new PB derivatives which could solve the problem of toxicity and poor solubility,so that to get some novel nti-inflammatory and immunomodulatory drugs.