Protective Effects And Mechanisms Of Sodium Ferulate Against Neurotoxicity Induced By Amyloid Beta-protein

ObjectiveAlzheimer's disease (AD) is a neurodegenerative disease. AD is characterized by an accumulation of extracellular deposits of amyloid - β protein ( Aβ ) and neurofibrillary tangles in the brain that is correlated with progressive cognitive decline and neuron loss. However, the underlying mechanisms of toxicity and activation of neuronal cellular signaling cascades induced by Aβ are not full-y understood. There is growing evidence that amyloid deposition provokes a mi-croglial - mediated inflammary response. Furthermore, Aβ - stimulated activation of microglia and astrocytes results in increase of proinflammatory cytokines and neuronal degeneration. Significantly, both epidemiological and clinical trial data have demonstrated the value of anti - inflammatory therapies for lowering the incidence, slowing the progression, and reducing the symptomatic severity of AD.The mitogen - activated protein kinase ( MAPK) signaling pathway spans from the plasma membrane to. the nucleus and serves to transmit extracellular signal from cell membrane receptors to their intracellular targets. The MAPK su-perfamily includes extracellular signal - regulated kinases ( ERKs) , Jun amino -terminal kinases (JNK1/2/3) , p38MAPK and ERK5. ERK pathway plays a major role in regulating cell growth and differentiation. JNKs and p38 MAPK are activated during various stress conditions such as oxidative stress, UV radiation and are considered to be pro - apoptotic pathways. Akt/protein kinase B ( PKB) is activated via a phosphoinositide 3 - kinase (PI3K) - dependent signaling pathway. Akt/PKB has received widespread attention as an important anti - apoptotic protein. Therefore, inhibition of Aβ -induced JNK and p38MAPK acti-vation and upregulation of ERKl/2 and Akt/PKB pathways might dedicate a new target in alleviation of AD.Sodium ferulate ( SF) , extracted from a traditional Chinese herbal medicine , has potent antioxidant and anti - inflammatory activities. It has recently been reported that long term administration of ferulic acid protects mice against learning and memory deficits induced by centrally administered 3 - amyloid. The primary site of action of ferulic acid could be microglia and astrocytes. In the present study, we investigated the MAPK and Akt/PKB signaling events in the inflammatory response and apoptosis evoked by preaggregated AfJ^ ₃5, and the protective effects of SF by the methods of Nissl staining, immunoeytochemi-cal staining, Western blotting, and RT - PCR in vivo and in vitro.Materials and MethodsAnimals and treatmentSixty Sprague - Dawley rats ( weighing 180 -200 g, Grade II , Certificate NO:2003 -0009, Experimental Animal Center of China Medical University) were randomly divided into six groups (each group 10 rats) : control, APjj.j^ A025-35 + ibuprofen (15 mg ? kg"1) , Ap-^j + SF at 50, 100, and 250 mg ? kg"1. The rats were administrated with SF or ibuprofen through ig for 3 weeks prior to AP25 -35 injection and 1 week after the injection. The rats were injected intracerebroventricularly with AP25-35OO (xL) or saline solution by means of a Hamilton microsyringe. Two days later, Morris water maze was used to measure the spatial learning and memory ability of rats. The rats were trained in Morris water maze test for 5 consecutive days following intracerebroventricular injection. On the sixth day, probe test were given. Seven days after injection, the CA1 region was microdissected for Western blotting, reverse transcription -polymerase chain reaction ( RT - PCR) ,and the measurement of caspase -3 activity. Animals (5 in each group) used for Nissl staining and immunocvtochemi-cal staining of glial fibrillary acidic protein ( GFAP) were anesthetized and perfused transcardially with 4% paraformaldelyde.Cell cultures and treatment of culturesTo determine whether SF has protective effect on neuronal apoptosis mediated by Af$25₃5 induced macrophages activation, macrophages were stimulated with Ap^.^in the presence or absence of SF( 100,500, and 1000 jxmol ? L"1) for 48 h and then the cell -free supernatant of Aj325₃5 stimulated macrophages was transferred to the culture of cortical neurons for 72 h. Neuronal apoptosis was quantitated by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst33258 staining. To determine the effects of SF on expressions of p38 MAPK, ERK1/2, Akt/PKB, and 70 kDa ribosomal protein S6 ki-nase ( p70S6K) , cultured macrophages were randomly divided into seven groups: control, Ap25₃5(15 jxmol ? L"1), A325₃5 +SF(100, 500, and 1000 jxmol ? L"1), Ap^.;+ PD98059 (10 jxmol ? L"1), Afiis-v + SB203580 (10 jxmol ? L"1) , and Afi^^ + LY294002 (10 fxmol ? L"1). Macrophages were pretreated with SF for 10 min or PD98059, SB203580 and LY294002 for 30 min prior to the addition of AP25₃5( 15 |xmol ? L^'). Protein expression of inducible nitric oxide synthase (iNOS) in macrophages was determined by im-munocytochemical staining. After the macrophages were stimulated for 0.5 or 2 h, macrophage extracts were prepared for Western blotting. In addition, the su-pernatants of cultured macrophages were collected for quantification of TNFa and NO.Results1. Protective effect of sodium ferulate on Afi^ -35 induced learning and memory dysfunction.Aj$25₃5 treated rats took longer time to find the platform than the control rats did. Treatment with SF (50, 100 and 250 mg ? kg"1) and ibuprofen (15 mg ? kg"1) for 4 weeks significantly shorten the escape latency to find the platform. Probe trial experiments revealed learning deficits in AP25-35 treated rats. SF significantly attenuated Ap^ ₃5 induced learning and memory damage in the water maze.2. Effect of sodium ferulate on A p^ ₃5 induced morphological change inhippocampal pyramidal neurons.The arrangement of hippocampal CAl pyramidal neurons of control group was clearly discernible. The arrangement of pyramidal neurons of AS group was sparse and the Nissl body was decreasing or dissolving. The number of pyramidal neurons in hippocampal CAl region was decreased. The arrangements of pyramidal neurons of AS + SF (50, 100 and 250 mg ? kg"1) groups were better than that of AS - treated group.3. Sodium ferulate inhibited astrocyte activation in rat hippocampusA625-35 resulted in infiltration of astrocytes in hippocampal CAl, as well as transformation of astrocytes from a resting to an activated state, highlighted by iphenotypic changes characterized by long, thick branching. SF at 50 mg ? kg"1, 100 mg "kg"1 and 250 mg ? kg"1 significantly inhibited the AB25₃5 -induced astrocytic reaction in hippocampal CAl.4. Sodium ferulate inhibited the A@25.35 -induced increase in expressions of IL - 18, TNFa, iNOS, and COX - 2 in rat hippocampusIntracerebrOventricular injection of preaggregated AB25.35 increased mRNA levels of IL -1B, TNFa, and iNOS and protein expressions of IL - 1B and COX -2 in rat hippocampus. SF (50, 100,and 250 mg ? kg"1) significantly inhibited AB25-35 induced these increases in protein and mRNA expressions in a dose - dependent manner. Ibuprofen (15 mg ? kg"1) showed a similar inhibition in AB^.as -induced increase of IL-1B, TNFa , iNOS, and COX-2 expressions.5. Sodium ferulate inhibited the ABj^s - induced increase in phospho -p38 MAPK and phospho - JNK1/2 expressionsIntracerebroventricular injection of preaggregated AB25₃5 led to a significant increase in phospho - p38 MAPK and phospho - JNK1/2 protein expressions. The ABy.ss - induced increase in activation of p38 MAPK and JNK1/2 was paralleled by an AB25 -35 induced increase in IL -1B protein expression. The AB25₃5 - induced increase in activation of p38 MAPK and JNK1/2 was prevented by SF (50 mg ? kg"1, 100 mg ? kg"1 and 250 mg ? kg"1, daily for 4 weeks) in a dose - dependent manner. Ibuprofen (15 mg ? kg"1) showed a similar inhibition in AB25 -35 induced increase in phospho - p38 MAPK andphospho - JNK1/2 expressions.6. Intracerebroventricular injection of A(325₃5 downregulated ERK1/2, Akt/protein kinase B and the effects of sodium ferulateSeven days after injection, AP25-35 elicited a*significant decrease in the phosphorylated form of ERKl and ERK2 compared with control rats. SF (50 mg ? kg"1, daily for 4 weeks) partly abolished the Ap25₃5 - induced decrease in ERKl and ERK2 activation, but SF (100 mg ? kg"1 and 250 mg ? kg"1, daily for 4 weeks) and ibuprofen (15 mg ? kg"1) did not prevent the decrease in phosphorylated ERKl and ERK2 induced by Ap25₃5.Treatment with Ap25₃5 also decreased the expression of phosphorylated Akt/PKB compared with control rats. SF (50, 100, and 250 mg ? kg"1, daily for 4 weeks) completely reversed the effect of Ap25₃5 on phosphorylated Akt/ PKB in a dose - dependent manner. Ibuprofen (15 mg ? kg"1) showed a similar effect in change of AfJ^.^ -induced phosphorylated Akt/PKB. However, no significant differences in the expression of total Akt/PKB were observed among treatment groups.7. Sodium ferulate decreased the A325-35 induced increase in expression levels of Fas ligand and p53 proteinWe assessed expressions of pro - apoptosis protein p53 and FasL. The results showed that AP25.35 significantly enhanced the expression levels of FasL and p53 protein in hippocampal CA1 regions. SF (50, 100 and 250 mg ? kg"1) and ibuprofen (15 mg ? kg" ) significantly inhibited the A(3 - induced increase in FasL and p53 protein expression levels in a dose -dependent manner.8. Sodium ferulate attenuated AJJ^ ₃5 - induced caspase cascades Intracerebroventricular injection of preaggregated AP25.35 led to the processing of inactive procaspase - 9 into their active forms. Procaspase - 9 proteol-ysis was confirmed by the increase of a 37 kDa fragment in hippocampal CA1. SF (50, 100 and 250 mg ? kg"1) and ibuprofen (15 mg ? kg"1) significantly prevented AP25-35 - induced procaspase -9 cleavage. According to the picture of the apoptotic pathway, caspase - 9 activity is responsible for procaspase — 3 and procaspase - 7 activation ( executioner caspases) by proteolytic cleavage. Ap25₃5 induced procaspase -3 processing and caspase -3 activation by the ap-pearance of pi9 fragments. Similarly, Casepase -7 was also cleaved to its pi9 active form in AB -treated rat hippocampus. SF (50, 100 and 250 mg ? kg"1) and ibuprofen (15 mg * kg ) significantly inhibited caspase - 3 and caspase -7 activation induced by AB. Caspase - 3 activity was measured by cleavage of the caspase - 3 substrate (Ac - DEVD - pN A ). Caspase - 3 activity was significantly enhanced in hippocampal CAl prepared from AB - treated rats compared with control rats. This AB - induced increase in caspase - 3 activity was inhibited by SF (50, 100 and 250 mg ? kg ) and ibuprofen (15 mg ? kg'1).9. Sodium ferulate reduced poly ( ADP - ribose) polymerase ( PARP) cleavageIntact PARP (116 kDa) expression was significantly lower in A625-35 treated animals compared with control animals and was associated with the appearance of the 89 kDa fragment of PARP. In SF - treated animals, the expression level of intact PARP (116 kDa) was higher while the expression of the 89 kDa fragment was lower when compared with that of AS - treated animals.10. Sodium ferulate protected cultured neurons from apoptosis mediated by AB25 -35 - induced macrophage activationThe supernatant of the macrophages stimulated with A 625.35 (15 jxmol ? L"') for 48 h resulted in a significant increase in percentage of apoptotic neurons with the nuclear stain Hoechst 33258 compared with control cultures. SF (0.1, 0.5, and 1 mol ? L" ) significantly suppressed neuronal apoptosis induced by supernatants from AB25₃5 - stimulated macrophages. Incubation of cultured macrophage with AB25₃5>for 48 h significantly increased TNFa and NO production. In addition, AB2s₃5activated multiple signaling pathways involving ERK1/2, p38MAPK, and phosphoinositide 3 - kinase (PD - K)/Akt, and PI3 - K/p70S6K pathways. AB25₃5 - induced increase of TNFa level in macrophages involves activation of ERK1/2. SF decreased these AB25 -35 - induced changes in dose - dependent manner.Conclusion1. Sodium ferulate improves AB25 -35induced learning and memory dysfunc-tion.2. p38 MAPK and JNKl/2 signal transduction pathways play the pivotal role in the inflammatory response and apoptosis evoked by AP25-35 in vivo. In addition, the data also show that downregulation of the survival signals ERK1/2 and Akt/PKB may contribute to the apoptosis of the cells.3. Sodium ferulate prevents AfJ^ ₃5 - induced neurotoxicity through suppression of p38 MAPK and JNKl/2 activation and upregulation of phospho -ERK1/2 and phospho - Akt/PKB in rat hippocampus.4. An in vitro injured neuronal model was established by using the toxicity of Ap^.35 -stimulated macrophages toward cortical cells. In this model, sodium ferulate exerted neuroprotective effects on cortical neurons via an inhibition of neurotoxic secretions from activated macrophages.