atherosclerosis , one of the main complications of diabetes , constitutes the leading cause of morbidity and mortality in today 's world .
macrophages , as major inflammatory contributors , are key modulators of atherosclerotic plaque formation and progression : m1 macrophages , which are related to inflammatory effects , stimulate inflammation and promote plaque progression , while m2 macrophages , which link with anti - inflammatory roles , contribute to inflammation resolution and atheroma regression [ 2 , 3 ] .
previous studies have demonstrated that excessive inflammatory m1 monocytes / macrophages emerge in peripheral blood of both prediabetic and diabetic patients [ 4 , 5 ] .
furthermore , our previous study also found that m1 monocytes / macrophages , the inflammatory subset , increased in circulation and atherosclerotic plaque in stz - induced diabetic mice and were related to enhanced inflammation and accelerated atherosclerosis .
thus , the increased inflammatory macrophages in diabetes contribute to persistent low grade inflammation and accelerated atherosclerosis .
various factors are responsible for macrophage inflammatory activation in diabetes . besides hyperglycemia , the advanced glycation end products ( ages ) , which are generated irreversibly in high glucose condition , are another group of critical pathogenic factors in diabetes .
because inflammatory macrophages dominate the progression of atherosclerosis , blockage or attenuation of ages - induced macrophage inflammatory response might alleviate diabetic atherosclerosis .
in addition to its antihyperglycemic effects , metformin , with over half - decade use as first - line therapy for type 2 diabetes , slows down diabetic atherosclerosis development   through mechanisms that are still not fully understood . recently , metformin 's anti - inflammatory properties were demonstrated in lipopolysaccharide induced macrophages [ 11 , 12 ] , and previous studies had shown that metformin could downregulate rage expression   and suppress nfb signaling in various cells [ 14 , 15 ] . as our previous work proved that rage / nfb signaling was involved in ages - induced macrophage inflammatory activation , it is reasonable to hypothesize that metformin might inhibit ages - induced inflammatory response in macrophages .
therefore , the present study tested the hypothesis and could provide novel elucidation for the antiatherosclerotic effect of metformin .
male c57bl/6 mice ( 8 weeks old ) were purchased from slac laboratory animal co. , ltd .
the mice were housed under specific pathogen - free conditions with controlled temperature ( 2225c ) and 12  h light / dark cycles ; they were given standard chow and water ad libitum .
all animal experiments were approved by the ethics committee of xinhua hospital affiliated to shanghai jiao tong university school of medicine ( approval number xhec - f-2016 - 012 ) .
mice were anesthetized with pentobarbital ( 50  mg / kg , i.p . ) to minimize suffering and killed by cervical dislocation without recovery from anesthesia .
tibias and femurs were removed and bone ends were cut off , and bone marrow was flushed with pbs supplemented with 1% fetal bovine serum ( fbs , gibco , australia , cat # 10099 - 141 ) .
a single cell suspension was prepared by filtering the cells through a 40  m strainer ( bd falcon , cat # 352340 ) .
the cell suspension was centrifuged at 1500  rpm for 5  min , and the cell pellet was resuspended in culture medium .
bone marrow cells were cultured for 7 days at 37c with 5% co2 in dulbecco 's modified eagle 's medium ( dmem ) , high glucose ( hyclone , beijing , china , cat # sh30022.01b ) supplemented with 10% fbs , 2  mm l - glutamine ( sigma - aldrich , missouri , usa , cat # 59202c ) , 100  u / ml penicillin and 100  g / ml streptomycin ( beyotime , jiangsu , china , cat # c0222 ) , and 20  ng / ml gm - csf ( peprotech , new jersey , usa , cat # 315 - 03 ) .
macrophages ( > 95% ) were identified by flow cytometry with anti - cd11b apc and anti - f4/80 fitc ( ebioscience , california , usa , cat # 17 - 0112 and cat # 11 - 4801 ) staining .
bmdms were seeded at a density of 0.5  10/ml and cultured overnight before stimulation .
based on experimental protocol , the bmdms were treated with different concentrations of metformin ( 0.25 , 1.0 , and 2.0  m ) or ages ( 200  mg / l ) for different time periods . for pathway studies
, the bmdms were pretreated with anti - rage antibody or ammonium pyrrolidinedithiocarbamate ( pdtc ) or compound c for 60  min .
the anti - rage neutralizing antibody ( r&d systems , minnesota , usa , cat # af1179 ) was reconstituted at 0.2  mg / ml in sterile pbs and further diluted with culture medium to the final concentration of 20  g / ml . metformin hydrochloride ( abcam , cambridge , uk , cat # ab120847 ) was dissolved in water at a concentration of 50  mm and further diluted with culture medium to the final concentrations ( 0.25 , 1.0 , and 2.0  m ) .
the nfb inhibitor pdtc ( abcam , cambridge , uk , cat # ab141406 ) was dissolved in dmso at a concentration of 100  mm and further diluted with culture medium to the final concentration of 50  m ( contains 0.5% dmso , v / v ) .
the ampk inhibitor compound c ( abcam , cambridge , uk , cat # ab120843 ) was firstly dissolved in dmso at a concentration of 10  mm and further diluted with culture medium to the final concentration of 5  m ( contains 0.5% dmso , v / v ) . to rule out the influence of dmso , proper dosages of dmso
were added to culture medium to meet the groups with the highest concentration of dmso .
age - bsa was purchased from anyan - bio technology ( shanghai , china , cat # ay-4710p ) , and the corresponding amount of bsa was used as control .
total rna was extracted from bmdms using trizol reagent ( takara , liaoning , china , cat # 9109 ) , and 1  g of total rna was reverse transcribed to cdna using the primescript rt master mix kit ( takara , liaoning , china , cat # rr036a ) . real - time pcr array analysis was performed by using the sybr premix ex taq kit ( takara , liaoning , china , cat # rr420a ) in a total volume of 20  l , with 2  l of cdna primers ( 0.2  mm each ) , 10  l of sybr green , and 0.4  l of rox dye ii .
the standard pcr conditions consisted of 95c for 30  sec , followed by 40 cycles of 95c for 5  sec and 60c for 34  sec , with a final dissociation stage ; the samples were run on an abi 7500 detector ( applied biosystems , california , usa ) .
the amounts of target genes were determined and normalized to the amount of gapdh cdna ( sangon biotech , shanghai , china , cat # b661304 ) .
the sequences of the primers ( synthesized by sangon biotech , shanghai , china ) for the target genes were as follows : fwd 5-ctcacaagcagagcacaagc-3 and rev 5-tccagcccatactttaggaaga-3 for il-1 ; fwd 5-tctgcaagagacttccatcca-3 and rev 5-agtctcctctccggacttgt-3 for il-6 ; fwd 5-ggtgcctatgtctcagcctc-3 and rev 5-ccacttggtggtttgtgagtg-3 for tnf- ; fwd 5-tgcactaccaaagccacaag-3 and rev 5-tgatcctcatgccagtcagt-3 for il-10 .
equal amounts of protein ( 4060  g ) were resolved on sds - page and transferred to polyvinyl difluoride ( pvdf ) membranes .
the blots were blocked with 5% milk in tbst for 1  h at room temperature and then incubated with primary rabbit anti - mouse antibodies overnight at 4c . the antibodies against p - ampk ( 1  :  1000 , cat # ab133448 ) and rage ( 1  :  1000 , cat # ab3611 ) were purchased from abcam ( cambridge , uk ) , and the antibodies against nfb-65 ( 1  :  1000 , cat # 8242s ) and p - nfb-65 ( 1  :  1000 , cat # 3033s ) were purchased from cell signaling technology ( massachusetts , usa ) .
the blots were subsequently incubated with the secondary goat anti - rabbit antibodies conjugated with horseradish peroxidase ( 1  :  1000 ) for one hour and enhanced using a chemiluminescence system ( chemidoc xrs+ , bio - rad laboratories , usa ) .
the intensity of each band was normalized to the loading control tubulin ( beyotime , jiangsu , china , cat # at819 ) .
after different stimulations , single cell suspensions of bmdms were prepared in flow buffer and incubated with antibodies .
anti - mouse cd86 pe ( ebioscience , california , usa , cat # 12 - 0862 ) was used to identify m1 macrophages while anti - mouse cd206 fitc ( biolegend , california , usa , cat # 141704 ) was used to identify m2 macrophages .
results were acquired with a bd canto ii flow cytometer ( bd biosciences , usa ) and analyzed by the flowjo software ( tree star , usa ) .
after different stimulations , cells were washed thrice with cold pbs and fixed in 4% ( w / v ) paraformaldehyde for 20  min and then washed with pbs again .
next , cells were incubated in buffered normal goat serum to prevent nonspecific binding of antibodies for 1  h at room temperature .
they then were incubated overnight with antibody against nfb-65 ( 1  :  200 , cat # 8242s ) purchased from cell signaling technology ( massachusetts , usa ) , followed by incubation with cy3 goat anti - rabbit igg ( 1  :  500 ; beyotime , cat # a0516 ) for 1  h at 37c .
dapi was used to stain the cell nuclei ( blue ) at a concentration of 1.43  m ( sigma , st .
one - way analysis of variance ( anova ) was used to assess the effects of one factor among multiple groups , and post hoc testing was done by tukey test .
two - way anova was used to assess the effects of two factors among multiple groups , and post hoc testing was done by bonferroni test .
chicago , usa ) for windows . a two - tailed value of p < 0.05 was considered statistically significant .
our previous study elucidated that ages promoted bmdms to express proinflammatory cytokines through rage / nfb signaling . to confirm this finding ,
here we tested cytokine expression profile of bmdms after ages stimulation with or without anti - rage neutralizing antibody or pdtc pretreatment .
bmdms were divided into 4 groups : control , ages , ages + anti - rage , and ages + pdtc group .
cells in the last two groups were pretreated with anti - rage antibody ( 20  g / ml ) or pdtc ( 50  m ) for 60  min , respectively , and then , together with ages group , the three groups were cultured with ages ( 200  mg / l ) for 24  h. the control group was treated with bsa ( 200 
mrna levels of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) and anti - inflammatory cytokine ( il-10 ) were measured by real - time pcr .  
figure 1 shows that ages markedly increased mrna expression of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) ( figures 1(a ) , 1(b ) , and 1(c ) ) , while only slightly upregulating that of il-10 ( figure 1(d ) ) , indicating that ages predominantly induced inflammatory response in murine macrophages .
in addition , pretreatment with anti - rage antibody or pdtc ameliorated the proinflammatory effects of ages ( figures 1(a ) , 1(b ) , and 1(c ) ) .
therefore , rage / nfb signaling is involved in ages - induced inflammatory response in macrophages . because metformin has anti - inflammatory potential
[ 11 , 12 ] , next we tested whether metformin inhibited ages - induced inflammatory response in macrophages .
bmdms were divided into 5 groups : control , ages , ages + met 0.25 ( metformin 0.25  m ) , ages + met 1.0 ( metformin 1.0  m ) , and ages + met 2.0 ( metformin 2.0  m ) .
cells in the last 3 groups were pretreated with different concentrations of metformin ( 0.25 , 1.0 , and 2.0  m ) for 60  min , respectively , and then , together with ages group , the 4 groups were stimulated with ages ( 200  mg / l ) for 24  h. the control group was treated with bsa ( 200 
mrna levels of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) and anti - inflammatory cytokine ( il-10 ) were measured again by real - time pcr .  
figure 2 shows that metformin dose dependently inhibited ages ' enhancement on mrna expression of proinflammatory cytokines ( figures 2(a ) , 2(b ) , and 2(c ) ) , while promoting that of il-10 ( figure 2(d ) ) , indicating that metformin inhibited ages - induced inflammatory response in bmdms . as the results showed that 2.0  m metformin had the strongest effects on downregulating genes expressions of proinflammatory cytokines but upregulating mrna expression of anti - inflammatory cytokine , we administrated 2.0  m metformin for the following experiments .
next , we tested whether inhibition of rage / nfb pathway was responsible for metformin 's suppressive effects on ages - induced inflammation . because metformin is an agonist of ampk and activation of ampk can abate inflammation in various cells [ 14 , 16 , 17 ] , we also evaluated activity of ampk .
firstly , bmdms were divided into 4 groups : control , ages , met , and ages + met group . in ages group , cells were cultured with ages at 200  mg / l for 24  h ; in met group , cells were cultured with metformin at 2.0  m for 24  h ; in ages + met group , cells were pretreated with metformin at 2.0  m for 60  min and then cultured with ages at 200  mg / l for 24  h ; in control group , cells were cultured with bsa at 200  mg / l for 24  h. western blot analysis was performed to measure protein levels of rage and phosphorylated ampk ( p - ampk ) .  
figure 3(a ) shows that ages significantly upregulated expression of rage and reduced levels of p - ampk , while metformin had the opposite effect .
moreover , pretreatment with metformin significantly attenuated effects of ages on rage upregulation and ampk inactivation .
then , we evaluated the effect of ages on activation of nfb pathway in macrophages with or without metformin ( 2.0  m ) pretreatment for 60  min before ages ( 200  mg / l ) stimulation for different time intervals ( 0 , 30 , 60 , and 180  min ) .
in western blot analysis ( figure 3(b ) ) , the p - p65/p65 ratio in the ages group markedly increased after ages stimulation , peaking at 60  min and decreasing thereafter , indicating that nfb was activated after ages stimulation .
furthermore , the p - p65/p65 ratio in the ages + met group was much lower at each time point than in the ages group , suggesting that ages - induced nfb activation was partly inhibited by metformin pretreatment . taken together , these findings suggested that metformin inhibited rage / nfb signaling . because metformin is an ampk activator , we next performed experiments to confirm whether metformin 's suppression of nfb signaling is ampk dependent .
bmdms were divided into 4 groups : control , ages , ages + met , and ages + met + cc group . because ages - induced nfb activation peaked at 60  min after ages stimulation , the duration of ages stimulation was set to 60 minutes . in ages group , cells were cultured with ages at 200  mg / l for 60  min ; in ages + met group , cells were pretreated with metformin for 60  min and then cultured with ages at 200  mg /
l for 60  min ; in ages + met + c - c group , cells were pretreated with compound c , an ampk inhibitor , at 5  m for 60  min , and then they were treated with metformin at 2.0  m for 60  min followed by ages at 200  mg /
l for 60  min ; in control group , cells were cultured with bsa at 200  mg / l for 60  min .
figure 4 shows that p65 nuclear translocation in ages group was significantly higher relative to control group indicating nfb activation ; metformin pretreatment significantly inhibited p65 nuclear translocation ; and compound c pretreatment abolished metformin 's effects .
subsequently , we tested whether metformin 's anti - inflammatory effect on ages - induced macrophages was dependent upon ampk activation .
bmdms were divided into 4 groups : control , ages , ages + met , and ages + met + c - c group . in ages group , cells were cultured with ages at 200  mg / l for 24  h ; in ages+met group , cells were pretreated with metformin for 60  min and then cultured with ages at 200  mg / l for 24 ; in ages + met + c - c group , cells were pretreated with compound c at 5  m for 60  min , and then they were treated with metformin at 2.0  m for 60  min followed by ages at 200  mg / l for 24  h ; in control group , cells were cultured with bsa at 200  mg /
mrna levels of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) and anti - inflammatory cytokine ( il-10 ) were tested again by real - time pcr .  
figure 5 demonstrated that pretreatment with compound c attenuated metformin 's inhibition on proinflammatory cytokines mrna expression and promotion of il-10 mrna expression in macrophages .
thus , these data indicated that the anti - inflammatory function of metformin was at least partly dependent on ampk activation .
m1 or m2 macrophages are , respectively , considered as pro- and anti - inflammatory macrophages . because cd86 is one of the important surface markers for m1 ( proinflammatory ) macrophages and cd206 for m2 ( anti - inflammatory ) macrophages , we next studied metformin 's impact on ages - induced surface markers ( cd86 and cd206 ) expression on macrophages by flow cytometry analysis .
bmdms were divided into 6 groups : control , ages , ages + anti - rage , ages + pdtc , ages + met , and ages + met + c - c . in ages group , cells were cultured with ages at 200  mg / l for 24  h ; in ages + anti - rage group , cells were pretreated with anti - rage neutralizing antibodies for 60  min followed by ages at 200  mg / l for 24  h ; in ages + pdtc group , cells were pretreated with pdtc for 60  min , followed by ages at 200  mg /
l for 24  h ; in ages + met group , cells were pretreated with metformin at 2.0  m for 60  min and then cultured with ages at 200  mg /
l for 24  h ; in ages + met + c - c group , cells were pretreated with compound c at 5  m for 60  min , and then they were treated with metformin at 2.0  m for 60  min followed by ages at 200  mg /
l for 24  h ; in control group , cells were cultured with bsa at 200  mg / l for the same amount of time .
m1 surface marker cd86 and m2 surface marker cd206 were detected by flow cytometry analysis . as expected , ages significantly increased cd86 but did not affect cd206 expression ; and pretreatment with anti - rage antibody or pdtc significantly abated ages ' effect .
pretreatment with metformin partly reversed ages ' effects on cd86 expression and markedly increased cd206 expression ; however , compound c abated metformin 's potency ( figure 6 ) .
the present study provides novel insight into a hypoglycemic agent : metformin inhibits the inflammatory response induced by advanced glycation end products in murine macrophages partly through ampk activation and suppression of rage / nfkappab signaling .
macrophage is a vital player in atherosclerosis , with its inflammatory response determining progression of atherosclerotic lesions .
ages are recognized as strong inflammatory mediators in the diabetic microenvironment , inducing an inflammatory response in macrophages [ 20 , 21 ] .
therefore , ages - induced macrophage activation and inflammation augmentation are critical mechanisms contributing to diabetic accelerated atherosclerosis , rendering it important to inhibit inflammatory response in macrophages in order to slow down or even block atherosclerotic progression .
recently , metformin was shown to have anti - inflammatory effects on lipopolysaccharide ( lps ) or pma induced macrophages [ 11 , 22 , 23 ] .
however , it is still unknown whether metformin was able to suppress ages - induced inflammatory response in macrophages .
the present study showed that pretreatment with metformin not only reduced mrna expression of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) but also upregulated mrna expression of anti - inflammatory cytokine ( il-10 ) in macrophages .
previous study has demonstrated that rage / nfb axis is important in driving inflammation in ages - induced macrophages . in the present study
, we found that blockade of rage or nfb signaling could significantly attenuate ages - induced genes expression of inflammatory cytokines , which confirmed the critical role of rage / nfb signaling in ages - induced inflammatory response .
in addition , although evidence is mounting that metformin downregulates rage expression or inhibits nfb activity in several cell types [ 1315 ] , until the present study , there had been no reports on metformin 's inhibition on ages - induced rage / nfb signaling in macrophages .
therefore , for the first time , we demonstrated that metformin inhibits ages - induced inflammatory response in macrophages via suppressing rage / nfb activation . of note
, in the present study , we found blockade of rage or nfb signaling or administration of different concentrations of metformin could not completely abolish the proinflammatory effects of ages .
such findings hint that pathways other than rage / nfb might also be responsible for ages - induced inflammatory response in macrophages . as is well known , in addition to rage
, ages can bind with several other receptors such as age - receptor complex , scavenger receptor , and toll - like receptor 4 ( tlr-4 ) to transmit messages [ 24 , 25 ] .
furthermore , besides nfb pathway , mapk and stat signaling have been proven to be implicated in driving inflammatory response in macrophages [ 26 , 27 ] .
therefore , rage / nfb signaling is just one of the many pathways mediating the inflammatory response induced by ages ; other potential pathways remain to be studied .
metformin is an ampk activator , and ampk signaling suppresses nfb activation thereby attenuating inflammatory responses [ 28 , 29 ] . in the present study , pretreatment with compound c , an ampk inhibitor , abated metformin 's suppression on nfb as well as proinflammatory cytokines expression , indicating that metformin 's inhibition of ages - induced inflammatory response in macrophages was ampk dependent . of note
, in the present study , ages slightly increased mrna expression of il-10 , while pretreatment with metformin significantly strengthened ages ' induction of il-10 mrna expression in a dose dependent manner . in light of the powerful anti - inflammatory function of il-10 , the promotion effect of metformin upon il-10 expression
furthermore , blockade of nfb signaling did not change il-10 expression while ampk blocking treatment significantly ameliorated metformin 's promotion on il-10 expression , indicating that ages - induced il-10 expression was nfb independent while metformin 's enhancement on expression of il-10 was at least partly ampk dependent .
therefore , metformin not only suppressed expression of proinflammatory cytokines through ampk signaling induced nfb inhibition but also enhanced expression of anti - inflammatory cytokine ( il-10 ) also via ampk activation .
a previous study found that metformin primed macrophages into different phenotypes based on the microenvironment . in the present study , metformin inhibited ages - induced m1 surface marker cd86 expression while increasing m2 surface marker cd206 expression .
based on the metformin 's effects on cytokines expression , results of the present study suggest that metformin probably inhibits ages - induced macrophage m1 polarization and might enhance macrophage m2 polarization . because macrophage polarization determines function , that is , pro- ( m1 ) or anti - inflammatory ( m2 ) [ 18 , 19 ] , the results further support metformin 's inhibition of ages - induced inflammatory response in macrophages .
ages - induced proinflammatory status of macrophages might be a critical mechanism responsible for diabetic accelerated atherosclerosis . therefore ,
the present study demonstrates that metformin inhibits ages - induced inflammatory response in murine macrophages through ampk activation and suppression of rage / nfb signaling , thereby providing a novel molecular mechanism responsible for metformin 's benefits on diabetic atherosclerosis .
 advanced glycation end products ( ages ) are major inflammatory mediators in diabetes , affecting atherosclerosis progression via macrophages . 
 metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated . 
 the present study of murine bone marrow derived macrophages showed that ( 1 ) ages enhanced proinflammatory cytokines ( interleukin-1 ( il-1 ) , il-6 , and tumor necrosis factor- ( tnf- ) ) mrna expression , rage expression , and nfb activation ; ( 2 ) metformin pretreatment inhibited ages effects and ages - induced cluster designation 86 ( cd86 ) ( m1 marker ) expression , while promoting cd206 ( m2 marker ) surface expression and anti - inflammatory cytokine ( il-10 ) mrna expression ; and ( 3 ) the ampk inhibitor , compound c , attenuated metformin effects . in conclusion , metformin inhibits ages - induced inflammatory response in murine macrophages partly through ampk activation and rage / nfb pathway suppression . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion