Protective Effects Of Active Fraction And Sparstolonin B On Inflammatory Injury Of Intracerebral Haemorrhage And 3D-QSAR And Molecular Docking Studies Of Sparstolonin B

A Chinese herb,Scirpus yagara Ohwi,is a perennial,aquatic plant grown in North and East China.It belongs to the family of Cyperaceae,whose tubers have long been used in traditional Chinese medicine for the treatment of postpartum abdominal pain,amenorrhea,and several inflammatory diseases.Previous studies conducted on this plant have revealed an abundant number of secondary metabolites,including trans-stilbenoids,triterpenoids,flavonids and phenolic acids.In addition,these constituents have demonstrated anti-inflammatory,anti-oxidant,anti-HIV and anti-cancer activity.Recently,an active fraction?AF?with anti-inflarmatory has been screened out from S.yagara through the experiments in vitro,and eight components have been identified by HPLC;Further studies found that AF significantly decreased TNF-? and IL-6 production induced by LPS in RAW264.7 macrophage and improved the survival rate of endotoxin shock mice.In addition,we have identified several compounds from the tubers,including methyl-3,6-dihydroxy-2-[2-?2-hydroxyphenyl?-ethynyl]benzoate,ferulic acid,sparstolonin B?SsnB?,sciryagarol ?,?,and etc.Among these compounds,SsnB,a selective Toll-like receptor 2?TLR2?and TLR₄ antagonist,can act directly on TLR2 and TLR₄ TIR domains or TIRAP/Mal protein,which is an indispensable adaptor bridging TLR2/TLR₄ TIR domain and MyD88 interaction,and blocked TLR2 or TLR₄ signaling cascade,decreased TNF-? and IL-6 production.Intracerebral haemorrhage is a stroke subtype that is associated with high mortality and morbidity,and patients who survive typically have major neuro logical impairments.Recent 20 years,this disorder accounts for 20-30%of strokes in Asia,with about 2 million cases worldwide per year.When ICH occurs,blood components including RBCs,leukocytes,macrophages,and plasma proteins such as thrombin immediately enter the cerebral parenchyma.The inflammatory response begins immediately after the presence of blood components in the parenchyma,and is characterized by accumulation and activation of inflammatory cells.The resident microglia and astrocytes are believed to be the early inflammatory cells in response to the extravascular blood components.In ICH,Thrombin and complements can activate microglia,possibly via protease-activated receptors?PARs?,hematoma components such as fibrinogen and heme induces microglial activation and downstream cytokine expression via the TLR₄ signaling pathway.Therefore,this paper studies the protective effects of AF and SsnB on mouse with ICH-induced secondary inflammatory injury and its mechanism.To study the anti-inflammatory effects of AF,25 ?Lvolume of autologous nonanticoagulated blood was collected from the tail artery of the mouse and then injected into the striatum using a syringe pump.Neurological deficits of the mouse was assessed 1.3 and 5 days after ICH using a 28-point neurological deficit scale,and brain water content of the mouse cerebral tissues after ICH was measured at third day;The concentrations of pro-inflammatory cytokines in perihematoma tissues.including interleukin-6,interleukin^-1? and tumor necrosis factor-?,were measured by ELISA assay.The results show that AF significantly reduced the neurological deficit scores and the brain water content after ICH.AF also markedly inhibited the levels of IL-6,IL-lp and TNF-a at first day and third day after ICH.Anti-inflammatory active fraction of Scirpus yagara can improve the brain injury in mouse induced by ICH.The mechanism may involve decreased expression of pro-inflammatory cytokines of mouse after ICH.To study the protective effects of SsnB on mouse with ICH,we investigated the effect of SsnB on neuronal cell death induced by Hb using a microglia-neuron trans-well system.Autologous nonanticoagulated blood was collected from the tail artery of the mouse and then injected into the striatum using a syringe pump.Neurological deficit of the mouse with ICH was assessed l,3,5 and 7 days after SsnB administration using modified neurological severity score system,and brain water content of the mouse cerebral tissues after ICH was measured at the same day;The concentrations of pro-inflammatory cytokines in perihematoma tissues,including interleukin-6,interleukin-lp and tUmor necrosis factor-a5 were measured by ELISA assay.The results show that SsnB significantly reduced the neurological deficit scores and the brain water content,and inhibited the levels of IL-6,IL^-1? and TNF-? at first day and third day after ICH.SsnB can improve the brain injury in mouse induced by ICH.The mechanism may involve increased the neuronal survival rate and decreased expression of pro-inflammatory cytokines of mouse afterICH.In order to obtain better ant?inflammatory compounds,a series of spanrstolonin B?SsnB?and its analogue derivatives were synthesized,and the n-octanol/water partition coefficient?Kow?of these derivatives was determined by the high performance liquid chromatography?HPLC?method.In addition,all compounds' anti-inflammatory activities of inhibiting the secretion of TNF-? and IL-6 in LPS-induced or Pam3csk4-induced RAW264.7 cells were evaluated Then we applied 3D-QSAR analyses using the comparative molecular field analysis?CoMFA?and comparative molecular similarity index analysis?CoMSIA?methods.According to the CoMFA model and CoMSIA model,we explained the structure activity relationship,and it indicated that hydroxyl group?s?at C-5' or?and?C-8 resulted in production of anti-inflammatory activities.Finally,Surflex-dock was employed to comparatively explore mechanism of the inhibitors when acting chemically with TLR₄.The results show the derivatives mainly interact with the sites of Arg 434,Val 435,Thr 436,Glu 437 and Asn 456 of mouse TLR₄/MD-2/LPS complex.And compounds SsnB,SanLeng diphenyllactone and its products of alkaline hydrolysis or isoquinolone at 0-2 position interact with TLR₄/MD-2/LPS complex better than the other compounds.This study can be conducive to gaining compounds with better activity and providing a theoretical basis for the better structural modification of the SsnB.