Neuroprotective Effects And Related Mechanisms Of Ultra Low Molecular Weight Heparin

ObjectiveNeurodegenerative disorders(NDDs)are chronic and progressive neurologic disorders.Since life expectancy has increased steadily in developed countries in the course of the 21st century,NDDs therefore become a rapidly major health problem. Although the precise cellular mechanisms underlying the pathogenesis of NDDs remain to be clarified,Ca²⁺overload in neurons is implicated in the pathogenesis of NDDs.Excessive elevation of Ca²⁺in neurons activates intracellular Ca²⁺-dependent signaling cascades that eventually lead to neuronal necrosis or apoptosis.In the present study,we focus on the following four issues:(1)Effect of ULMWH on cultured cortical cells.(2)Effect of ULMWH on glutamate(Glu)-induced apoptosis in cultured cortical cells;(3)Effect of ULMWH on intracellular Ca²⁺concentrations([Ca²⁺]_i)both in brain cells isolated from new-born rats and in cultured cortical cells;(4)Effect of ULMWH on inositol 1,4,5-trisphospate(IP₃)-induced intracellular Ca²⁺release.The aim of the present study is to elucidate the neuroprotective effect and possible molecular mechanisms of ULMWH,and to supply effective treatment options for NDDs.Methods1.Cortical cells of fetal rats were cultured in vitro and treated with Glu,sodium azide,KCl and caffeine,respectively,to mimic the pathogenesis of NDDs.The viability of cortical cells and the activity of lactate dehydrogenase(LDH)in culture medium were measured,respectively.2.Primary cultures of rat cortical cells were performed in vitro and treated with Glu to induce apoptosis.Hoechest 33258 staining,flow cytometry(FCM)with FITC-Annexin V/PI dual staining and DNA fragmentation were employed to evaluate neuronal apoptosis,respectively.Mitochondrial transmembrane potential(MMP)was determined by rhodamine 123 FCM.Bcl-2 protein,Bax protein,cytochrome c and cleaved caspase-3 were measured by Western blot,respectively.3.Using Fura-2/AM,a Ca²⁺sensitive fluorescent indicator,to load the brain cells isolated from new-born rats or primary cultured rat cortical cells,the intracellular free Ca²⁺concentration([Ca²⁺]_i)was measured with Fura-2/AM fluoremetry.4.Brain cells were isolated from new-born rats.Endoplasmic reticulum(ER)or mitochondria(Mit)was prepared from cell suspension by subcellular fractionation and sucrose density gradient centrifugation.By loading Fura-2/ AM into the organelles,the IP₃-induced Ca²⁺release from intracellular Ca²⁺stores was directly examined.The effect of ULMWH on IP₃-induced Ca²⁺release was investigated.Results1.Effect of ULMWH on damaged cortical cells(1)Effect of ULMWH on cortical cells damaged by Glu Incubation with 100μmol·L^-1Glu for 24 h significantly decreased the cell viability and remarkably increased the LDH activity in cortical cells,indicating that the cortical cells can be damaged by excessive Glu under this experimental condition.Theses changes in cell viability and LDH activity induced by Glu were reversed by pretreatment with ULMWH,indicating that ULMWH can protect cortical cells against Glu-induced damage.(2)Effect of ULMWH on cortical cells damaged by sodium azide Incubation with 64 mmol·L^-1sodium azide for 4 h caused a significant decrease of cell viability and a remarkable increase of LDH activity in cortical cells,suggesting that the cortical cells can be damaged by sodium azide under this experimental condition.Theses changes in cell viability and LDH activity induced by Glu were reversed by pretreatment with 1.0 mg·L^-1ULMWH,indicating that ULMWH can protect the cortical cells against sodium azide-induced damage.(3)Effect of ULMWH on cortical cells damaged by KCl or caffeine Incubation with 200 mmol·L^-1KCl for 20 min or 10 mmol·L^-1caffeine for 30 min caused a significant decrease of cell viability and a remarkable increase of LDH activity in cortical cells,suggesting that the cortical cells can by damaged by KCl or caffeine under this experimental condition.Pretreatment with ULMWH attenuated neither the decrease of cell viability nor the increase of LDH activity induced by KCl or caffeine,indicating that ULMWH can not exert protective effects on KCl or caffeine-damged cells.2.Effect of ULMWH on glutamate-induced apoptosis in cortical cells(1)Morphological changes visualized by Hoechst 33258 staining Cells are considered apoptotic if they show irregular,shrunk,and condensed nuclei with bright fluorescence.Quantification of the Hoechst 33258 stained apoptotic cells indicated that about 19.9%of control cells exhibited apoptotic morphology,whereas about 33.21%of Glu-treated cells had apoptotic-like appearance,which had significant difference compared with that in control cells.In ULMWH-treated cells,the proporation of apoptosis was about 19.45%,which had no significant difference compared with that in control cells.The percentage of apoptosis in ULMWH pretreated cells was decreased to about 20.47%,which had significant difference compared with that in Glu-treated cells,however,no significant difference compared with that in control cells,indicating that ULMWH pretreatment can decrease the number of apoptosis induced by Glu.(2)Dual parameter flow cytometry analysis Cortical cells were stained with fluorescein isothiocyanate(FTTC)labeled annexin-V,and simultaneously with PI stain,to discriminate intact cells(annexin^-/PI^-)from apoptotic cells(annexin⁺/PI^-)and necrotic cells(annexin⁺/PI⁺).In control cells,3.69%of cortical cells were undergoing typical apoptosis.As compared with control cells,the percentage of apoptosis in 100μmol·L^-1Glu-treated cells was 20.91%,indicating that Glu can induce neuronal apoptosis.However,the proportion in ULMWH(0.01,0.1 and 1 mg·L^-1)-pretreated cells was decreased to 12.7%,7.01%,and 6.13%,respectively,indicating pretreatment with ULMWH can decrease significantly the percentage of apoptosis induced by Glu.(3)DNA fragmentation and caspase-3 expression After exposure of cortical cells with 100μmol·L^-1Glu for 24 h,DNA electrophoresis showed a typically pronounced DNA ladder of apoptosis,and Western blot showed a 56%increase in caspase-3 protein.However,DNA ladder and caspase-3 expression was significantly inhibited in 1 mg·L^-1ULMWH-pretreated cells,respectively.There was no significance in caspase expression between control cells and ULMWH-treated cells.(4)Bcl-2 and Bax expression in cortical cells As compared with control cells, treatment of cortical cells with 100μmol·L^-1Glu for 24 h up-regulated highly Bax protein level,down-regulated obviously Bcl-2 level,and decreased accordingly the ratio of Bcl-2:Bax.However,such changes were reversed completely by 1 mg·L^-1 ULMWH-pretreatment.There was no significance in the levels of Bax or Bcl-2 between ULMWH-treated cells and control cells.(5)Mltochondrial membrane potential(MMP)assay In control cells,Rh123 fluorescence intensity in Mit was about 1483.17,whereas in Glu-treated cell it was decreased to about 84.44(approxiamately 5.87%of control cells),indicating that Glu can induce mitochondrial membrane depolarization and damage Mit.After pretreatment with ULMWH(01,0.1 and 1 mg·L^-1),Rh123 fluorescence intensity was decreased to 285.66,593.13 and 847.07(approxiamately 20.12%,39.97%and 57.93 %of control cells),respectively,indicating that ULMWH can inhibit markedly mitochondrial membrane depolarization induced by Glu and protect Mit against Glu damage.(6)Cytochrome c release 100μmol·L^-1Glu exposure for 24 h resulted in a significant increase in Cyt C immunoreactivity in the cytosolic fraction compared with that in control cells.ULMWH pretreatment significantly attenuated the Glu-induced increase in Cyt C immunoreactivity in the cytosolic fraction compared with that in Glu-treated cells.There was a corresponding decrease in Cyt C immunoreactivity in the mitochondrial fraction in response to Glu compared with that in control cells.ULMWH pretreatment significantly raised the Glu-induced decrease in Cyt C immunoreactivity in the mitochondrial fraction compared with that in Glu-treated cells.3.Effect of ULMWH on intracellular calcium concentration in cortical cells(1)Effect of ULMWH on[Ca²⁺]_i in brain cells isolated from new-born ratsâ…°)Effect of ULMWH on resting[Ca²⁺]_i In the presence of extracellular Ca²⁺of 1.3 mmol·L^-1,the resting[Ca²⁺]_i in rat neuronal cells was 137.68±10.36 nmol·L^-1, which was markedly decreased in ULMWH(0.1 and 1 mg·L^-1)pretreated cells.In a Ca²⁺-free d-Hanks'solution containing 1 mmol·L^-1EGTA,[Ca²⁺]_i in rat neuronal cells was 97.97±9.44 nmol·L^-1,which was decreased markedly in ULMWH(0.1 and 1 mg·L^-1)pretreated cells,indicating that ULMWH induced the decrease of resting [Ca²⁺]_i may be attributable to suppressing intracellular Ca²⁺release.â…±)Effect of ULMWH on Gin-induced[Ca²⁺]_i elevation In the presence of extracellular Ca²⁺of 1.3 mmol·L^-1,100μmol·L^-1Glu increased the[Ca²⁺]_i by 93.7% to 267.92±6.49 nmol·L^-1.Preincubation with ULMWH(0.1 and 1 mg·L^-1)decreased significantly the[Ca²⁺]_i elevation induced by Glu,there was a significant difference compated with that in Glu-treated cells.In a Ca²⁺-free d-Hanks'solution containing 1 mmol·L^-1EGTA,the rise of[Ca²⁺]_i induced by Glu was still observed,however,the increase of[Ca²⁺]_i was much lower than that in Hanks'solution containing Ca²⁺of 1.3 mmol·L^-1,indicating that the rise of[Ca²⁺]_i induced by Gin may be partially attributed to intracellular Ca²⁺release.When 1 mg·L^-1ULMWH was added into Ca²⁺-free d-Hanks solution prior to the application of Glu,the rise of[Ca²⁺]_i induced by Glu was completely suppressed and there was a significant difference compared with that in Glu-treated cells,indicating that ULMWH decreased the rise of[Ca²⁺]_i in isolated brain cells induced by Glu may be ascribed to suppressing intracellular Ca²⁺release.â…²)Effect of ULMWH on KCl-induced[Ca²⁺]_i elevation In a Ca²⁺-free Hanks solution containing 1 mmol·L^-1EGTA,KCl 200 mmol·L^-1did not affect the[Ca²⁺]_i in cortical cells,indicating that KCl can not induce intracellular Ca²⁺release.However, high K⁺ increased the[Ca²⁺]_i by 80%to 248.66±10.67 nmol·L^-1in d-Hanks solution containing Ca²⁺of 1.3 mmol·L^-1,indicating that KCl can induce extracellular Ca²⁺ influx.Although the elvation of[Ca²⁺]_i induced by high K⁺ was decreased slightly in ULMWH(0.01,0.1 and 1.0 mg·L^-1)-pretreated cells,there was no significant difference compared with that in KCl-treated cells,indicating that ULMWH can not inhibit extracellular Ca²⁺influx.â…³)Effect of ULMWH on caffeine-induced[Ca²⁺]_i elevation In the presence of extracellular Ca²⁺of 1.3 mmol·L^-1,10 mmol·L^-1of caffeine increased the[Ca²⁺]_i to 248.36±10.67 nmol·L^-1.Although the elvation of[Ca²⁺]_i induced by caffeine was decreased slightly in ULMWH(0.01,0.1 and 1.0 mg·L^-1)-pretreated cells,there was no significant difference compared with that in caffeine-treated cells,indicating that ULMWH can not inhibit caffeine-mediated intracellular Ca²⁺release.(2)Effect of ULMWH on Glu-induced[Ca²⁺]_i elevation in cultured cortical cells As compared with control cells,Glu-treated cells showed a significant rise of [Ca²⁺]_i,which was partially inhibited by pretreatment with 1 mg·L^-1ULMWH,In Ca²⁺-free medium containing 1 mmol·L^-1EGTA,the rise of[Ca²⁺]_i induced by Glu was still observed,however,the increase of[Ca²⁺]_i was much lower than that in medium containing Ca²⁺,indicating that the rise of[Ca²⁺]_i induced by Glu was partially attributable to intracellular Ca²⁺release.When 1 mg·L^-1ULMWH was added into Ca²⁺-free medium prior to the application of Glu,the rise of[Ca²⁺]_i induced by Glu was completely suppressed.Additionally,the magnitude of[Ca²⁺]_i decreased by 1 mg·L^-1ULMWH in medium containg Ca²⁺was almost equal to that in Ca²⁺-free medium,indicating that ULMWH decreased the rise of[Ca²⁺]_i induced by Glu in cortical cells may be ascribed to suppressing intracellular Ca²⁺release.4.Effect of ULMWH on inositol 1,4,5-trisphosphate-induced intracellular Ca²⁺release from Ca²⁺stores(1)Effect of ULMWH on IP₃-induced intracellular Ca²⁺release from ER Addition of 10μmol·L^-1IP₃ could cause a significant increase of Ca²⁺in ER suspension from 200.14±6.89 nmol·L^-1to 292.43±12.04 nmol·L^-1,indicating that a release of Ca²⁺from ER can be triggered by IP₃,which was significantly inhibited by pretreatment with ULMWH(0.01,0.1 and 1 mg·L^-1).When pretreated with 1 mg·L^-1 ULMWH,the inhibitory percent of Ca²⁺release reached about 60.3%.(2)Effect of ULMWH on IP₃-induced intracellular Ca²⁺release from Mit Addition of 10μmol·L^-1IP₃ could cause a significant increase of Ca²⁺in Mit suspension from 198.76±8.79 nmol·L^-1to 198.86±7.09 nmol·L^-1,indicating that a release of Ca²⁺from Mit can be triggered by IP₃,which was significantly inhibited by pretreatment with ULMWH(0.01,0.1 and 1 mg·L^-1).When pretreated with 1 mg·L^-1 ULMWH,the inhibitory percent of Ca²⁺release reached about 57.29%.Conclusions1.ULMWH can significantly not only increase cell viability but also decrease LDH activity in primary cultured cortical cells induced by GLu or sodium azide, indicating ULMWH can exert neuroprotective effect on cortical cells damaged by Glu or sodium azide.2.ULMWH can markedly prevent the Glu-induced apoptotic morphological change,decrease the percentage of apoptosis in Glu-treated cells,up-regulate the expression of Bcl-2,down-regulate the expression of Bax,decrease caspase-3 activation and inhibit Cyt C release into cytosol,suggesting that ULMWH can attenuate apoptosis in cortical cells induced by Glu. 3.ULMWH can attenuate the resting[Ca²⁺]_i in Ca²⁺-free d-Hanks solution in isolated brain cells and the increase of[Ca²⁺]_i induced by Glu in Ca²⁺-free medium in cortical cells,which may partially be attributed to suppressing intracellular Ca²⁺ release from internal stores.4.ULMWH can directly and remarkably suppress IP₃-induced intracellular Ca²⁺ release from ER and Mit.