Study On The Immunosuppressive Activity Of Pestalotiopyrone M,a Product Derived From Mangrove Microorganisms,and Its Mechanism Of Targeting CN/NFAT Signaling Pathway

Immunosuppressants are drugs that can suppress the body’s abnormal immune response.At present,it is widely used in hypersensitivity diseases,organ transplantation and autoimmune diseases.Autoimmune diseases include rheumatoid arthritis,lupus erythematosus,dermatomycosis,inflammatory bowel disease,and immune hemolytic anemia,etc.,all of which can be treated with immunosuppressive agents.Calcineurin（CN）is a serine/threonine specific phosphatase that relies on Ca²⁺calmodulin for signal transduction.CN is considered calmodulin binding protein,not only to adjust a variety of physiological functions,such as activation of T cells and is associated with some autoimmune diseases.The discovery of cyclosporin A（Cs A）and FK506,which are immunosuppressants of CN target enzymes,provides a basis for the screening of natural product immunosuppressive drugs.Because Cs A and FK506 have greater toxic and side effects,it is important to find highly effective and low-toxic immunosuppressants from natural products.Mangroves are unique coastal forest ecosystems distributed in tropical and subtropical regions of the world.Due to the harsh environment of low oxygen,high salinity and high temperature,mangrove with unique adaptive ability and the development of secondary metabolic pathway structure may be produced a variety of secondary metabolites,the function of these metabolites is the source of a variety of biological activity,plays an important role in medicine.Mangrove endophytic fungi are the second largest group of marine fungi,which contain structurally rich secondary metabolites and have a variety of pharmacological activities.Mangrove endophytic fungi point out a new direction for the development of lead compounds.A variety of secondary metabolites are isolated from mangrove endophytic fungi,which can produce anti-inflammatory,antibacterial,anti-tumor and immunosuppressive effects.Therefore,mangrove endophytic fungi are an important resource for natural medicine research and development.In this study,p NPP was used as a substrate to test the calcineurin activity,and the mangrove-derived products were screened for immunosuppressive activity.Four monomeric compounds of the secondary metabolites of the endophytic Aspergillus fumigatus of the R.mucronata were found,five compounds of secondary metabolites of the endophytic Phomosis asparagi of the Rhizophora mangle and one compound of secondary metabolites of endophytic fungus Pestalotiopsis sp of the Rhizophora stylosa have immunosuppressive activity.The new pyrone compound Pestalotiopyrone M（PyM）isolated from the endophytic fungus Pestalotiopsis sp HHL101,a Chinese mangrove plant Rhizophora stylosa,can effectively inhibit calcineurin activity and exhibit a dose-dependent inhibitory effect.Among the three mangrove microorganism-derived monomers,PyM is the least toxic to normal lymphocytes,has significant immune activity,and is the first new compound discovered in the world.Therefore,in this study,we selected PyM with low toxicity and significant immunosuppressive activity for subsequent immunosuppressive research.This study aimed to study the immunosuppressive activity of PyM in mouse lymphocytes and its mechanism of targeting calcineurin-related NFAT signaling pathway.Previous studies have shown that PyM has significant inhibitory activity on calcineurin(IC₅₀ is 19.67±0.08μM),and it shows low cytotoxicity to splenocytes(IC₅₀ is 385.78±17.90μM).PyM can effectively inhibit the proliferation of lymphocytes induced by Con A(IC₅₀ is 46.77±0.68μM),indicating that PyM can effectively inhibit the proliferation of T lymphocytes and B lymphocytes.In addition,PyM also has an inhibitory effect on the unidirectional mixed lymphocyte response(IC₅₀ is 42.67±1.00μM),indicating that the compound can also inhibit immune rejection.We used PI（propidium iodide）staining to detect the cell cycle distribution and found that PyM can inhibit the cell cycle of splenocytes from G1 phase to S phase and G2 phase,indicating that PyM can inhibit mitosis and cell proliferation of mouse lymphocytes.Annexin V-FITC/PI double staining showed that after PyM acts on mouse spleen cells,the proportion of early and late apoptotic cells increases,indicating that PyM induces cell apoptosis in a concentration-dependent manner.The CN/NFAT pathway is one of the most important signal pathways related to immunity.Immunofluorescence confirmed that PyM inhibits the nuclear transfer of NFAT1 protein on the NFAT signal pathway.Reverse transcription PCR and real-time quantitative PCR experiments show that PyM also exerts an inhibitory effect on IL-2 m RNA in the NFAT pathway.we further used Western Blot experiments to prove that PyM significantly inhibits the expression of NFAT1 protein.Finally,we used the ELISA method to also confirm that PyM exerts an inhibitory effect on the cytokines related to the NFAT pathway,and has an inhibitory effect on IL-2,IL-4,TNF-α,INF-αhave different degrees of inhibition.These results indicate that PyM uses CN enzyme as the target enzyme to participate in the immunosuppressive effect of the NFAT signaling pathway.Therefore,PyM can be used as a potential immunosuppressive agent,which provides the possibility for the development and utilization of natural sources of high-efficiency and low-toxicity immunosuppressive agents.