Screening Of In Vitro Neuroprotective Activity Of Special Environment Microbial Natural Products And Their Mechanism Research

Neonatal hypoxie-ischemic encephalopathy(NHIE)refers to hypoxic ischemic injury of the brain caused by asphyxia during perinatal period.NHIE not only seriously threatens the lives of newborns,but is also one of the most common causes of sick children after the neonatal period NHIE causes human neonatal death and chronic neurological dysfunction.Astrocytes are involved in the blood-brain barrier,and they are the first damaged brain cells to suffer from ischemic risk.Protecting damaged-astrocytes is an effective strategy for the treatment of NHIE Therefore,based on the occurrence mechanism of NHIE,it is of important practical significance to find a reliable neuroprotective drug for curing neonatal encephalopathyIn recent year,the possibility of discovering new compounds from terrestrial plants and environmental microorganisms had become smaller and smaller.Therefore,more and more researchers have begun toendophyte and marine microorganism.Special-environment microorganisms are readily to pay attention to microorganisms from special environment,especially produce special secondary metabolites with structural and biological diversity,which provide abundant lead compounds for developing new drugsOxygen-Glucose Deprivation(OGD)induced cell model is always used to minic NHIE in drug research.So,the OGD-damaged rat primary astrocytes model was established to mimic NHIE in vitro in this dissertation.The experiment showed that the apoptosis rate of astrocytes was over 50%after 6 h of hypoxia and hypoglycemia,indicating that OGD-induced cell model was established successfully for 6 h of hypoxia in the sugar-free medium.Then,the OGD-induced astrocyte model was used to screen the in vitro neuroprotective activity of natural products from endophyte M.roridum,endophyte C globosm and marine fungus P.janthinellum,and the related structural modification products in the dissertation.The result showed that NBS-1(8)was toxic to normal astrocytes significantly,while NaBH4-1(13),NaBH4-2(14),BnNCO(16)and guxi-2(17)showed a certain toxicity at concentrations greater than 20 ?g/mL.The left compounds had no toxicity to normal astrocytes.The following in vitro neuroprotective activity evaluation showed that cytoglobosin Ab(1),chaetoglobosin F(6),mCPBA-1(7),HWE-1(11),guxi-2(17),Penicilon B(18)and 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-3-methyl-2H-pyran-2-one(19)could significantly reverse OGD-induced death of rat astrocytes,revealing their protective effect to OGD-damaged astrocytes.The result showed that these compounds have a good potential in developing drugs for diseases related to ischemia and hypoxia injury.Based on the screening result,in vitro neuroprotective mechanism of active compounds chaetoglobosin F and guxi-2 was further explored in the dissertation.Chaetoglobosin F was one of cytochalasin compounds isolated from C.globosum.Chaetoglobosin F was found to reverse the OGD-promoted increase of LDH level and ROS activity,and decrease of SOD activity,indicating that it could reduce cell lysis and reduce oxidative stress.At the same time,it was found that chaetoglobosin F pretreatment up-regulated the expression of SIRT1 in the nucleus and reduced the expression of substrate proteins Hif-la and Ac-p53 of SIRT1,indicating the enhancement of SIRT1 deacetylase activity.NF-?B entered into the cytoplasm from the nucleus.The apoptotic rate of model astrocytes was decreased,along with the down-regulated expression of Bax,Caspase-3 and Cleaved caspase-3,and up-regulated expression of Bcl-2,suggested that the neuroprotective effect of chaetoglobosin F was associated with the prevention of apoptosis.In addition,it was also showed that chaetoglobosin F pretreatment could promote the growth of major neurites in neurons co-cultured with astrocyte after hypoxia,indicating that chaetoglobosin F could avoid the effect of astrocyte overactivation on neurons.The series of studies revealed that SIRT1 might be activated by chaetoglobosin F,then acted on the SIRTl/acety-p53/NF-?B pathway,leading to the reducing of OGD-induced oxidative damage and astrocyte apoptosis.Guxi-2 was a new cyclo-decapeptide compound isolated from endophyte M.roridum in A.anua.Guxi-2 was found to down-regulate the levels of LDH and ROS,and up-regulate the activity of SOD in OGD-induced cells,indicating that it could reduce cell death and alleviate the oxidative stress.After pretreatment with guxi-2,in OGD-induced cells,SIRT1 was expressed in the nucleus and its expression was up-regulated,while the levels of substrate proteins Hif-la and Ac-p53 were decreased,indicating the increasing of SIRT1 deacetylase activity.In addition,NF-?B entered into the cytoplasm from the nucleus and the expression of total NF-?B mRNA was decreased.Finally,the decreased expression of Bax,Caspase-3 and Cleaved caspase-3,and the increased expression of Bcl-2 indicated that the neuroprotective effect of guxi-2 was related to the prevention of apoptosis.A series of results suggested that guxi-2 exhibited significant neuroprotective activity.SIRT1 might be activated by guxi-2,then acted on the SIRT1/acety-p53/NF-KB pathway,resulting in the reducing of oxidative damage and astrocyte apoptosis induced by OGDSIRT1 negatively regulates oxidative stress and apoptosis-associated transcription factors through deacetylation(an epigenetic modification method),which likely become a new target for the clinical treatment of NHIE,Both these compounds are potential activators of SIRT1,which exerted significant neuroprotective activity by acting on SIRT1 to regulate the SIRT1/acety-p53/NF-KB pathway.They are promising drug lead compounds for curing NHIE.