Fisetin Improves Neural Protection And Functional Recovery In Rat Model Of Spinal Cord Injury

BackgroundSpinal cord injury(SCI)is a very serious and disabling disease in central nervous system(CNS)without effective treatment.After SCI,patients often suffer from sensory and motion function disorders,urinary retention or abnormal defecation,which imposes a heavy burden on society and patients.Nowadays,The pathogenesis and treatment of SCI is currently one of the hottest areas of medical research.Astrocytes are the most abundant glial subtype in CNS and play important roles in interacting with neurons and communicating with other neural cell types.Due to local micro-environment changes after SCI,astrocytes proximal to lesion zones become reactive.The process of reactive astrocytosis involves massive morphological changes and variaty of gene expression,contributing to glial scar formation,which is a major impediment to axonal regeneration.Overcoming this physical and biochemical barrier could be crucial for axonal regeneration and functional compensation during the progression of SCI.Fisetin is a dietary flavonoid commonly found in fruits and vegetables,but it is actually quite rare and present at low to very low concentrations in all of the sources.It has extensive physiological and pharmacological activities,including antioxidant,anti-aging,anti-inflammatory,anti-neoplastic and neuroprotective effects.Studies have also shown that fisetin enhances behavioral performance and attenuates reactive gliosis and inflammation during aluminum chloride-induced neurotoxicity.Furthermore,fisetin suppresses oxidative and neuroinflammatory responses in microglial cells.However,the effects of fisetin on reactive astrocytosis remain unclear.This study is divided into two parts as following:Part one Fisetin regulates astrocyte migration and proliferation in vitro1.ObjectiveUsing scratch-wound model,to explore the effect and underlying mechanism of fisetin on astrocyte migration and proliferation in vitro.The early results can set up a foundation for studing the effect of fisetin on glial scar formation and SCI,thus find a new therapy for SCI.2.Methods2.1 Cell culturePrimary astrocyte cultures were prepared from 1-day-old Sprague-Dawley rat brains,as described previously.Briefly,the cerebral hemisphere was freed of the meninges and cut into 1 mm3 cubes in Dulbecco's Modified Eagle Medium(DMEM).The tissue was dissociated by vortex mixing for 60 s,and the cell suspension was passed through 70 ?m and 20 ?m sterile mesh nylon filters.A volume of cell suspension containing about 4.5×105 cells was seeded in a 35 mm2 Falcon tissue culture dish.Primary astrocytes were cultured in DMEM containing 10%fetal bovine serum(FBS)and 0.15%penicillin-streptomycin reagent at 37? with 5%CO2.The medium was changed twice a week.Cultures of at least 4 weeks old were used for experiments.The primary cultured astrocytes was identified by GFAP immunofluorescence,and the rate of positive cells is more than 95%.2.2 Scratch-wound assayThe capacity of astrocytes to migrate was tested by scratching confluent astrocyte monolayers to induce natural cell migration,as reported previously.Astrocyte monolayers were wounded by dragging a sterile 200 ?L pipette tip across the surface.The detached cells and debris were removed immediately with three washes of phosphate-buffered saline(PBS).Cells were then maintained for an additional 24 h in culture medium with 1%FBS and different concentrations of fisetin(25?M and 50?M)or 0.1%DMSO as the control group.Images of the closing wound were acquired under an inverted microscope and analyzed using NIS Elements D 3.2.2.3 Cell apoptosis analysisAstrocytes were pretreated with fisetin(12.5 ?M,25 ?M,and 50 ?M)or 0.1%DMSO as the control group for 24 h.Then,cells were digested with 0.125%EDTA-free trypsin and collected by centrifugation at 2,000 rpm for 5 min.Cells were washed twice with PBS,resuspended in 500 ?L binding buffer,and incubated in the dark for 10 min at room temperature with 5 ?L of Annexin V-Fluor 488 and 5 ?L of propidium iodide(PI).The samples were assessed by flow cytometry.2.4 Click-iT EdU testAstrocytes were treated simultaneously with different concentrations of fisetin(12.5 ?M-50 ?M)or 0.1%DMSO as the control group and 5-ethyl-2'-deoxyuridine(EdU)(10?M),using the Cell-LightTM EdU Apollo567 In Vitro Flow Cytometry Kit for 24 h.The cells were then stained according to the following protocol:cells were collected by centrifugation at 1,500 rpm for 5 min,4%paraformaldehyde was added at room temperature for 30 min to fix the cells,followed by washing with PBS,then addition of 0.5%Triton X-100 for 15 min,two washes with PBS,and incubation with click reaction buffer(100 mM Tris-HCl,pH 8.5;1 mM CuSO4;100 ?M Apollo 550 fluorescent azide;and 100 mM ascorbic acid)for 30 min in the dark,washing with 0.5%Triton X-100,and final addition of 500 ?L PBS for the flow cytometry analysis within 1 h.2.5 Cell cycle assayAstrocytes were synchronized by starved for 12 h in serum free medium and treated subsequently with different concentrations of fisetin(12.5 ?M-50 ?M)or 0.1%DMSO as the control group for 24 h before analysis.Cell cycle analysis was performed by PI staining.Astrocytes were trypsinized and washed with ice-cold PBS(pH 7.4)and fixed in ice-cold 75%ethanol for more than 2 h.The cells were then washed with PBS,treated with 100 ?L RNase A at 37? for 30 min,and finally stained with 400 ?L PI in the dark at 4? for 30 min.Flow cytometry analysis was then carried out within 1 h.The analysis was performed using a cell cycle detection kit.2.6 Western blot analysisAstrocytes were treated simultaneously with different concentrations of fisetin(12.5 ?M-50 ?M)or 0.1%DMSO as the control group for 24 h before analysis.Then we conducted Western Blot to detect the protein expression changes of p-paxillin/paxillin,p-FAK/FAK,cyclinDl,p-Akt/Akt,p-Erk/Erk,p-p38/p38.We applied Quantity-One software to examine the gray value(GV)of WB bands.3.Results3.1 Effect of fisetin on astrocyte apoptosiswe measured the effect of fisetin on astrocyte cell apoptosis using flow cytometry analysis.Fisetin,at concentrations ranging from 12.5-50 ?M,did not significantly affect cell apoptosis after treatment for 24 h.3.2 Fisetin suppressed astrocyte migration.Fisetin significantly reduced the ability of astrocytes to migrate into the empty space.Control astrocytes reduced the gap at 24 h,while the gap was larger at 24 h with fisetin-treated group.Examination of the leading edge of the migrating cells revealed multiple protrusions and extensions in control astrocytes,but a relatively uniform flat edge in astrocytes in the fisetin group.To gain further insight into the effects of fisetin on migration of primary cultured astrocytes,we examined the expression of p-FAK and p-paxillin in vitro.Western blotting shows that both p-FAK and p-paxillin were decreased.Together,these results indicate that fisetin suppressed astrocytes migration.3.3 Fisetin inhibited astrocyte proliferation and cell cycle.Astrocyte proliferation was detected using EdU as a marker of dividing cells.The proportion of EdU(+)cells was markedly reduced in the fisetin-treated group compared with the control group,in a dose-dependent manner.Similarly,fisetin increased the percentage of cells in G1/G0 phase and decreased the percentage in G2 and S phase compared with the control group,as demonstrated by flow cytometry,and also in a dose-dependent manner.Accordingly,western blotting shows that fisetin also downregulated cyclinDl protein expression.Together,the data indicated that pretreatment with fisetin inhibited astrocyte proliferation and arrested the cell cycle at the G1 phase.3.4 Fisetin suppressed the Akt/Erk signaling pathway.In the present study,western blotting analysis showed that fisetin downregulated the phosphorylation of Akt and Erk in a dose-dependent manner,but had no effect on total levels of Akt and Erk.Furthermore,fisetin had no effect on the expression of p38 mitogen-activated protein kinase(p38MAPK;p38)or p38 phosphorylation levels compared with the control group.4.ConclusionIn summary,our study indicates that fisetin inhibits astrocytes migration in a scratch-wound model and causes cell cycle arrest in G1 which inhibits astrocyte proliferation via PI3K/Akt-and Erk-dependent signaling pathways,and further decreases glial scar formation.Fisetin may therefore have a potential application in the treatment of glial scar formation in SCI.Part twoFisetin improves neuron protection and functional recovery in rat model of spinal cord injury1.ObjectiveTo explore the effect of fisetin on neuron protection and functional recovery in rats after SCI and find a new potential drug for treating.2.Methods2.1 Establishment of SCI modelModified Allen's method was applied to conduct the SCI model on adult male SD rats(specific pathogen-free grade,weighing 220-250 g),the dorsal surface of the cord at T10 was impacted using a lOg weight dropped from a height of 25 mm to produce contusive SCI.Appearance of symptoms such as spinal cord edema and hemorrhage,delayed paralysis of both lower limbs,wagging reflection and urine retention confirmed the successful establishment of SCI rat model.2.2 Animal groups and treatmentA total of 126 rats were randomly divided into 3 groups:sham group(n=42),SCI group(n=42),fisetin-treated group(n=42).Fisetin-treated group received intraperitoneal injection of 25 ?mol/kg dose of fisetin(dissolved in 1%DMSO,0.9%saline),every 12 h until 7 days after surgery.The sham and SCI group were injected with 1%DMSO solution(dissolved in 0.9%saline),every 12 h until 7 days after surgery.2.3 Behavioral testUsing BBB(Basso,Beattie,and Bresnahan)scores,rats were forced to walk in an open area for 20 min at respectively 1 d,3 d,7 d,14 d,21 d and 28 d after surgery,and their hind limb movement was observed.2.4 Biochemical analysisTo evaluate the antioxidative effect of fisetin,biochemical kits were applied to measure superoxide dismutase(SOD)and lipid peroxidation end product malondialdehyde(MDA).All procedures were performed as the protocol of the kit strictly.2.5 LuminexThe serum level of interleukin(IL)-1?,IL-4,IL-6,IL-10 were measured at Id,3 d,7 d after surgery.The injured spinal cord was homogenized in saline with phenylmethyl sulfonyl fluoride(PMSF)and the assessment for all cytokines was performed using a commercial colorimetric kit according to the protocol strictly.All determinations were performed in duplicate serial dilutions.2.6 ImmunohistochemistryThe rats were sacrificed and fixed transcardially with ice-cold PBS and 4%PFA at day 3 and 28 post SCI.The spinal cord tissues containing the contusion epicenter were drawn and cut horizontally with eight-micrometer sections serially.Separate sections were incubated for rabbit anti-IL-1? antibody(1:200),rabbit anti-IL-4 antibody(1:200),rabbit anti-IL-6 antibody(1:200),rabbit anti-IL-10 antibody(1:200),mouse anti-Ibal(1:100),mouse anti-GFAP antibody(1:100),mouse anti-NF200 antibody(1:200)and mouse anti-?-tubulin ? antibody(1:800)at 4?.Overnight,the specimens were then washed with PBS and incubated with secondary antibody conjugated with horseradish peroxidase for 1 hour,then were stained with 4',6-diamidino-2-phenylindole(DAPI).The samples were photoed in 10 and 20 microscopic fields.2.7 Neural electrophysiological examinationNeural electrophysiological examination was used to detect SEP and MEP at day 28 post surgery.3.Results3.1 Fisetin promoted behavioral function recovery after SCIUsing BBB scores to evaluate the motor recovery of hindlimb at 1 d,3 d,7 d,14 d,21 d,28 d post surgery,fisetin can significantly improve BBB scores compared to SCI group(p<0.01),and ameliorate the condition of hematuria,urinary retention.3.2 Fisetin suppressed oxidative stress response in rats after SCIBy detecting the activity of SOD and concentration of MDA at 3 d,7 d post surgery,the activity of SOD in fisetin-treated group was significantly higher than SCI group,on the contrary,the concentration of MDA in fisetin-treated group was significantly lower than SCI group.The results indicated that fisetin can accelerate the expression of SOD and inhibit the production of MDA,thus suppress oxidative stress response in SCI.3.3 Fisetin inhibited inflammation after SCIBoth luminex and immunohistochemistry results showed that the level of pro-inflammatory cytokines IL-1?,IL-4,IL-6 in fisetin-treated group was downregulated compared to SCI group.On the contrary,the anti-inflammatory cytokine IL-10 was upregulated by fisetin.3.4 Fisetin inhibited microglia/macrophage activationBoth immunohistochemistry and Western Blot results indicated that the specific marker of microglia/macrophage Ibal was downregulated in the fisetin-treated group,3.5 Fisetin inhibited glial scar formationWe detected the biomarker of astrocytes via immunohistochemistry and found that the expression of GFAP was significantly downregulated in the fisetin-treated group.3.6 Fisetin promoted neuronal axon regeneration and repairWe detected the biomarker of neuronal axon via immunohistochemistry and found the expression of NF-200,?-tubulin III were significantly downregulated in the fisetin-treated group.Fisetin can promote neuronal axon regeneration and repair.3.7 Fisetin enhanced function recovery of nerve conductionCompared to SCI group,fisetin can shorten the potential time of both SEP and MEP.Fisetin can also enhance the amplitude of evoked potentials including both SEP and MEP.The result of nerve electrophysiological examination demonstrated that fisetin enhanced function recovery of nerve conduction in rat SCI model.4.ConclusionFisetin can improve neural protection and functional recovery after SCI through suppressing oxidative stress response,inflammation,microglia/macrophage activation,glial scar formation and promoting neuronal axon regeneration.