folic acid belongs to the vitamin b complex . it is vital for red blood cells and for many other cells in the body .
folic acid is also known as folate , folacin , vitamin b9 , vitamin m , folvite , acifolic , folcidin , and scientifically as pteroylglutamic acid .
the form of folic acid occurring naturally in food is called  folate  and it is a water - soluble vitamin .
previous studies have described anti - inflammatory effects exerted by folic acid , although the mechanisms underlying these actions are still not yet fully clarified [ 2 , 3 ] .
it was reported that combined supplementation of micronutrients , folate , and vitamin b12 has a beneficial effect in reducing inflammation in pregnancy , acting on the levels of inflammatory cytokines .
folate and vitamin b supplementation has been assessed as a potential clinical intervention in vascular disease [ 5 , 6 ] .
in addition , the administration of folic acid to subjects with primary arterial hypertension caused a decreased concentration of inflammation markers ( hscrp , icam-1 , and vcam-1 ) .
kolb and petrie reported that folate deficiency enhances the proinflammatory cytokine output of raw264.7 monocytes , suggesting that a folate deficiency may exacerbate cardiovascular disease by augmenting proinflammatory signals in the monocyte - macrophage lineage .
microglia , immune cells resident in the central nervous system ( cns ) , are responsible for monitoring the cns environment protecting neurons from invading microorganisms , as well as removing cell degradation products . after recognizing specific pathogen - associated patterns they initiate both innate and adaptive immune responses by producing an array of proinflammatory cytokines , free radicals , and nitric oxide ( no ) .
these microglia responses bring about pathogen clearance and tissue regeneration , but the resulting inflammatory state , if overactivated , may cause neuronal injury .
it is now accepted that neuroinflammation is an important contributor to neurodegeneration in various cns diseases .
suppressors of cytokine signaling ( socs ) proteins are intracellular proteins that inhibit cytokine signaling in a wide variety of cell types .
in particular , socs proteins have been found to be expressed by immune cells , as well as by cells of the cns , including microglia [ 11 , 12 ] . in the context of the inflamed cns
they are involved in preventing excess inflammation , protecting the brain tissue from severe inflammation - induced tissue damage .
the anti - inflammatory effects of folic acid in microglia cells have not yet been well described .
here we reported the effects of folic acid on lps - induced inflammatory responses of bv-2 microglia cells .
the regulatory mechanisms that cause the upregulation of socs1 and socs3 expression were also investigated .
the murine - microglia cell line was obtained from the american type culture collection ( manassas , va , usa ) .
cells ( 2  10  cells / ml ) were plated onto 24-well plates and incubated overnight in dulbecco 's modified eagle 's medium ( dmem ; invitrogen ) containing supplementary 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin , 100  g / ml streptomycin , and 2  mm - glutamine , ( life technologies - invitrogen , milan , italy ) at 37c in a 5% co2 humidified atmosphere at 37c . then
, the cells were treated with folic acid ( sigma - aldrich , milan , italy ) at final concentrations of 5 , 10 , 30 , 50 , 70  g / ml for 1 hour and then stimulated with lps ( from escherichia coli 055:b5 ; sigma - aldrich ) ( 1  g / ml ) for the time indicated in the different assays .
cell viability was determined by an mtt ( sigma - aldrich ) assay . in brief , bv-2 microglial cells ( 2  10  cells / ml ) were plated onto 24-well plates , submitted to folic acid treatment and incubated overnight .
then , culture media were carefully removed and 100  l of 0.5  mg / ml mtt in cell culture medium was added to each well and incubated for 4  h. finally , 100  l of 10% sds and 0.01  m hcl solution were added to each well to dissolve the formed formazan crystals .
the cell viability was calculated according to the following formula : % cell viability = [ od ( 560  nm ) tested compound / od ( 560  nm ) control cells ]  100 . in controls ,
after 24 hours of incubation , supernatants of cultured cells were collected and assayed for no production using griess reagent .
briefly , the samples were mixed with an equal volume of griess reagent ( carlo erba , milan , italy ) and then incubated at rt for 10 minutes .
the absorbance of supernatants was spectrophotometrically measured at 540  nm and the no concentration was calculated by extrapolation from a standard curve of sodium nitrite ( nano2 ) . to avoid interference by nitrites possibly present in the medium , a blank of each experiment
total rna was isolated from cells using trizol reagent according to the manufacturer 's protocol .
the mrna levels of various genes were quantified using the sybr green quantitect rtpcr kit ( roche , south san francisco , ca , usa ) .
data were analyzed using the relative standard curve method according to the manufacturer 's protocol .
the mean value of each gene after gapdh normalization at the time point showing the highest expression was used as calibrator to determine the relative levels of tnf- , il-1 , inos , il-10 , arginase ( arg)-1 , and cd206 at different time points .
after treatment as previously described , cells were washed twice in pbs , detached with ice - cold pbs , collected , and centrifuged at 600  g for 10  min .
the supernatants were then removed and the pellet was harvested and lysed by ice - cold lysis buffer [ 1% ( v / v )
triton x-100 , 20  mm tris - hcl , 137  mm nacl , 10% ( v / v ) glycerol , 2  mm edta , 1  mm phenylmethylsulfonyl fluoride ( pmsf ) , 20  m leupeptin
hemisulfate salt , and 0.2  u / ml aprotinin ( all from sigma aldrich ) ] for 30  min on an ice - bath .
the protein concentration in the supernatant was spectrophotometrically determined by bradford 's protein assay and the lysate was subjected to sds - page ( nupage electrophoresis system , invitrogen ) .
after electrophoresis , the resolved proteins were transferred from the gel to nitrocellulose membranes using iblot dry blotting system a ( life - technologies ) .
antibodies directed against -actin , inos , erk 1/2 , p - erk 1/2 , p - ib , ib , p38 mapk , p - p38 mapk , jnk , p - jnk , socs1 , and socs3 were all obtained from santa cruz biotechnology , inc .
( santa cruz , heidelberg , germany ) ( diluted 1  :  1000 for p - ib , erk 1/2 , p - erk 1/2 , p - p38 mapk , p38 mapk , jnk , p - jnk ; 1  :  100 for inos ; 1  :  500 for socs1 and socs3 , and 1  :  10,000 for -actin ) .
membranes were incubated with the secondary antibody horseradish peroxidase ( hrp ) conjugate ( santa cruz biotechnology ) diluted 1  :  2000 , for 60  min at room temperature in the dark on a shaker .
finally , bands were visualized by the chemiluminescent method ( biorad laboratories , hercules , ca , usa ) .
the bands obtained after immunoblotting were submitted to densitometric analysis using i d image analysis software ( kodak digital science ) , expressing results expressed as arbitrary units .
for elisa assay , the supernatant of cultured cells stored at 70c was used to measure the cytokines release .
the concentrations of il-1 , il-10 , and tnf- were determined by elisa according to the manufacturer 's protocols ( r&d system inc . ,
student 's t - test and analysis of variance ( one - way anova ) were performed on the results of at least five independent biological replicates .
none of the 550  g / ml range concentrations of folic acid tested affected the viability of bv-2 microglial cells , although folate 70  g / ml resulted to be cytotoxic ( table 2 ) .
thus , for the study of microglial cells functional responses we used concentrations ranging from 550  g / ml , as reported in literature .
folate treatment significantly lowered the lps - induced no release by microglia cells ( figure 1(a ) ) .
we also found that folate significantly and dose - dependently attenuated no production after 24  h lps treatment , reaching a maximal inhibition at 50  g / ml . interestingly , inos expression was significantly and dose - dependently downregulated in the presence of folate ( figure 1(b ) ) .
the qpcr analysis confirmed these results also for the mrna levels of this inflammatory mediator ( figure 1(c ) ) . taken together , these results indicate that folate treatment suppresses the release of no in lps - stimulated bv-2 microglial cells by modulating inos expression also at transcriptional levels .
rt - pcr was performed to determine whether folic acid regulates proinflammatory cytokine and microglia activation markers expression at transcriptional level . as shown in figure 2(a ) we demonstrated a marked increase in tnf- and il-1 mrna in bv-2 microglial cells after 6  h of lps treatment . by contrast a significant reduction of arg-1 and cd206 microglia activation markers was evidenced in lps treated bv-2 microglial cells .
no effect of treatment with folic acid alone on proinflammatory gene expression was observed in cells , proinflammatory cytokines mrna levels being similar in all tested concentrations ( 550  g / ml ) and comparable to the levels detected in control cells . interestingly , we observed that mrna expression of proinflammatory cytokines was significantly decreased in lps - treated cells after pretreatment with folic acid in comparison to microglial cells stimulated with lps alone , suggesting that , in lps - activated cells , folic acid was able to downregulate proinflammatory cytokine mrna expression , in a dose - dependent manner , reaching a maximal reduction at 50  g / ml ( figure 2(a ) ) .
folic acid treatment alone had no effect on the production of il-1 and tnf- in bv-2 microglia .
the increased levels of il-1 and tnf- levels in supernatants obtained from 24  h lps - stimulated bv-2 microglia resulted significantly reduced after pretreatment with folic acid in a dose - dependent manner , as shown in figure 2(b ) . concerning the effect of folate on the regulation of the anti - inflammatory cytokine , il-10 , in lps treated cells we observed a significant increase of il-10 , while folate pretreatment was able to upregulate this expression . interestingly , il-10 levels resulted significantly increased in folate pretreated cells in terms of both transcript and protein and this regulation was dose - dependent , as reported in figures 2(a ) and 2(b ) .
same results were revealed for the arg-1 and cd206 mrna of lps treated bv-2 microglial cells pre - treated with folic acid ( figure 2(a ) ) . the role played by folic acid in cell signalling induced by 12 
h lps stimulation was also investigated . for this purpose , we firstly investigated nf-b activation . since the degradation of ib- is essential for the nuclear translocation of nf-b p65 , we determined the effect of folic acid on lps - induced phosphorylation of the ib- protein , by western blotting . cells stimulated with lps exhibited a significantly increased p - ib expression in comparison to controls ( figure 3 ) .
densitometric analysis revealed a faint phosphorylation of ib in unstimulated cells ( figure 3 ) .
pretreatment with folic acid significantly dose - dependently reduced p - ib- in lps - activated cells , as reported in figure 3 .
in addition to the nf - kb pathway , the effect of folic acid on the activation of the erk 1/2 , jnk , and p38 pathways was examined in lps - activated microglia cells using western blotting analysis . as shown in figures 4(a ) and 4(b ) , folic acid significantly increased lps - induced phosphorylation of p38 kinase in bv-2 cells in a concentration - dependent manner , whereas jnk phosphorylation was dose - dependently reduced by folic acid ( figures 4(a ) and 4(d ) ) .
conversely , erk 1/2 kinases phosphorylation was not affected by folic acid treatment ( figures 4(a ) and 4(c ) ) . finally , the amounts of total erk 1/2 , jnk , and p38 were unaffected by lps in combination with folic acid treatment .
these results suggest that folic acid could modulate lps - induced inflammatory mediatory production through regulating multiple signalling pathways . to confirm the role of p38 on il-10 overproduction by 24  h lps activated cells , pretreated with folic acid ( 50  g / ml )
, the cytokine expression levels were analyzed by elisa . as shown in figure 5 , treatment with the p38 pharmacological inhibitor , sb203580 ( 10  m ) , induced a significant decrease of il-10 levels with respect to both lps - stimulated cells in presence of folate and cells treated with folic acid alone , highlighting the important role of p38 kinase in the upregulation of il-10 production by folic acid treatment .
conversely , lps treatment determined a significant downregulation of the expression levels of socs proteins in bv-2 cells in comparison to control , according to that reported in literature .
cell treatment with folic acid alone increased , in a dose - dependent manner , both socs1 and socs3 expression levels in bv-2 cells .
interestingly , folic acid pretreatment before lps stimulation was able to prevent the lps - induced inhibition , as reported in figure 6(a ) .
finally , the qpcr analysis revealed a significant increase of socs1 and socs3 transcripts in lps - activated bv-2 cells submitted to folate pretreatment ( figure 6(b ) ) .
in this study we show , for the first time , that folic acid pretreatment was able to polarize proinflammatory responses to anti - inflammatory responses in lps - activated microglia cells , blocking the activation of nf-b and jnk and upregulating p38 mapk phosphorylation .
the modulation of signaling pathways may also be related both to a potent inhibitory effect of folic acid on the expression of inos , as well as of no , il-1 , and tnf- production induced by lps , and to an upregulating action on anti - inflammatory cytokine il-10 release in activated microglia .
finally , we also demonstrated that folic acid was able to dose - dependently upregulate socs proteins expression in microglia cells .
the hallmark of neuroinflammation is the activation of microglia and the production of cytokines and inflammatory mediators including no , tnf- , il-1 , il-6 , and inf- , which can trigger neuronal damage [ 9 , 1517 ] . in the substantia nigra of parkinson 's diseases patients
an increased density of microglial cells expressing inos has been observed , which is considered responsible for no derived neuronal toxicity [ 19 , 20 ] .
in addition , proinflammatory cytokines , such as tnf- , il-1 , and inf- , are described to cause a potent activation of inos . in this context , the deletion of inflammatory inos using gene targeting has been shown to have a neuroprotective effect in mptp - treated mice [ 22 , 23 ] .
interestingly , our findings demonstrated that folic acid treatment significantly inhibited in a dose - dependent manner no , tnf- , and il-1 production in lps - activated bv-2 cells . in addition , reduced no production was dose - dependently modulated by folic acid through a downregulation of inos expression .
several intracellular signal molecules are involved in the regulation of the inflammatory responses , such as the mapks , a group of serine / threonine protein kinases comprising three subfamilies : the p42/p44 erks , jnks , and the p38 [ 24 , 25 ] .
mapk signaling pathways regulate a variety of cellular activities such as proliferation , differentiation , apoptosis , survival , and inflammatory responses [ 26 , 27 ] .
mapks can be activated by various extracellular molecules , such as lps , leading to the activation of transcription factors , including nf - kb which orchestrates the induction of many inflammatory cytokines [ 2830 ] . in this
regard , our results showed that folic acid was able to dose - dependently downregulate jnk phosphorylation in lps - stimulated cells .
similar effects have been reported on raw264.7 macrophages , in which folic acid treatment inhibited lps - stimulated jnk phosphorylation , resulting in the inhibition of proinflammatory responses .
intriguingly , our results showed that p38 phosphorylation resulted enhanced by folic acid treatment in a dose - dependent manner .
considering the importance of mapk signaling in the regulation of inflammatory responses we proceeded to investigate the role of p38 in the il-10 overregulation after folic acid treatment of lps - activated microglia cells .
for this purpose , we examined the effect of the p38 pharmacological inhibitor , sb203580 , and observed how il-10 production was significantly reduced in folic acid treated cells , suggesting a crucial role for p38 phosphorylation in the il-10 modulation . in this context , the p38 pathway has been reported to be important for il-10 induction in tlr - activated antigen - presenting cells   as well as in lps - stimulated b cells .
il-10 is a potent anti - inflammatory cytokine able to inhibit the production of endotoxin - inducible proinflammatory cytokines such as tnf- , il-1 , and il-6 in mononuclear phagocytes .
it has been shown that il-10 interferes with the activation of nf - kb , and this activity has been correlated with its ability to block the synthesis of other proinflammatory cytokines .
then , it is possible to postulate that , in our in vitro model , folic acid treatment of bv-2 cells determined a p38-dependent increase of il-10 , which in turn was able to block nf - kb activation .
il-10 has also been shown to induce the expression of socs1 and socs3 and this upregulation is thought to be one of the mechanisms by which il-10 mediates its downstream effects [ 11 , 35 ] .
moreover , it has been reported that il-10 induces the expression of socs3 in monocytes .
in addition , forced expression of the socs3 gene in myeloid cell lines markedly inhibits cytokine - induced activation of the jak / stat signaling pathway , indicating that il-10 may inhibit the production of proinflammatory cytokines , as observed in monocytes .
socs1 and socs3 have received increasing attention due to their pivotal roles in regulating immune responses , which result from the fatal effects of a deficiency of either protein [ 37 , 38 ] .
it has been reported that both socs1 and socs3 directly bind nf-b p65 , leading to its proteolysis and the suppression of nf-b activation [ 3941 ] .
interestingly , socs1 and socs3 have also been shown to regulate m1 and m2 macrophage polarization [ 42 , 43 ] . in this regard
, it was observed that socs1 is necessary to limit the m1 phenotype in response to ifn-/lps stimulation .
socs1-deficient mice resulted hypersensitive to lps signaling , as shown by an enhanced ib phosphorylation demonstrating the role of socs1 as a negative regulator of tlr-4 signaling . similarly to m1 macrophage , microglia cells also respond to insults by transiently activating the release of tnf- , no , and other pro - inflammatory mediators in order to eliminate pathogenic insults .
brain barrier , leukocyte recruitment , a leukocyte and lymphocyte influx , and typical hallmarks of neurodegenerative diseases [ 47 , 48 ] . to ensure wound healing
, microglia must rapidly return to a resting m2-like state , attenuating the inflammatory response and assuring homeostasis .
m2-polarized microglia secrete anti - inflammatory factors , including il-10 , as well as expressing socs proteins .
failure to shift to a m2 microglia phenotype or a predominance of m1-polarized microglia is associated with worsening tissue damage and neuronal loss , suggesting that m2-polarized microglia act as gate - keepers against neuroinflammation [ 46 , 4951 ] . in vivo
socs1 overexpression in oligodendrocytes suppresses deleterious actions observed in experimental autoimmune encephalomyelitis , as well as attenuating socs1 expression in microglia - promoted proinflammatory m1-like microglia and worsening neuroinflammation , suggesting that socs1 plays a crucial role in modulating cytokine responses and might be a promising therapeutic modulator in cns disease states .
socs3 expression resulted enhanced in rat cerebral cortex following a cortical impact injury and in the hippocampus following lithium - pilocarpine - induced seizure [ 53 , 54 ] . these observations underline the importance of socs1 and socs3 , among the socs family members , in regulating innate immune responses in cns .
although in literature a number of reports have described the anti - inflammatory effects of folic acid , there is a paucity of information regarding the modulation of socs proteins by folic acid in microglia cells [ 8 , 13 ] .
our study demonstrated that folic acid significantly attenuated the release of proinflammatory mediators in lps - activated microglia , blocking nf-b , and overregulating il-10 dependent socs proteins expression through p38 pathways ( figure 7 ) .
thus , we propose that upregulation of socs1/3 expression by folate may be an integrated part of the regulatory machinery of inflammatory responses in activated microglia and that at least two distinct pathways may lead to this result .
further in vivo studies of this activity could be useful to investigate the mechanisms involved in the anti - inflammatory actions of folate in order to permit their full exploitation and further explore their promise as therapy in neuroinflammatory diseases .
 we investigated the ability of folic acid to modulate the inflammatory responses of lps activated bv-2 microglia cells and the signal transduction pathways involved . to this aim 
 , the bv-2 cell line was exposed to lps as a proinflammatory response inducer , in presence or absence of various concentrations of folic acid . 
 the production of nitric oxide ( no ) was determined by the griess test . 
 the levels of tumor necrosis factor - alpha ( tnf- ) , interleukin-1 beta ( il-1 ) , and il-10 were determined by elisa . 
 inducible no synthase ( inos ) , nuclear transcription factor - kappa b ( nf-b ) p65 , mapks protein , and suppressors of cytokine signaling ( socs)1 and socs3 were analyzed by western blotting . tnf- and il-1 , as well as inos dependent no production , 
 resulted significantly inhibited by folic acid pretreatment in lps - activated bv-2 cells . 
 we also observed that folic acid dose - dependently upregulated both socs1 and socs3 expression in bv-2 cells , leading to an increased expression of the anti - inflammatory cytokine il-10 . 
 finally , p - ib , which indirectly reflects nf-b complex activation , and jnk phosphorylation resulted dose - dependently downregulated by folic acid pretreatment of lps - activated cells , whereas p38 mapk phosphorylation resulted significantly upregulated by folic acid treatment . 
 overall , these results demonstrated that folic acid was able to modulate the inflammatory response in microglia cells , shifting proinflammatory versus anti - inflammatory responses through regulating multiple signaling pathways . 