rheumatoid arthritis ( ra ) is a systemic autoimmune disorder with progressive articular damage that may result in lifelong disability [ 1 , 2 ] .
it typically causes a symmetrical chronic arthritis that causes joint pain , swelling and in some cases a systemic illness [ 3 , 4 ] .
the central pathogenesis of rheumatoid arthritis ( ra ) is characterized by not only marked hyperplasia of the fibroblast - like synoviocytes ( flss ) in response to the production of autocrine , but also paracrine molecules produced by infiltrating mononuclear cells [ 3 , 5 ] .
the aberrant hyperplastic flss display properties of transformed cells and destroy periarticular bone and cartilage . actually , flss actively participate in the invasive processes of ra . since flss proliferate abnormally
, they produce high levels of destructive enzymes and cytokines to decrease the susceptibility to spontaneous and induced apoptosis .
thus , it is important to elucidate the mechanisms of flss proliferation and apoptosis for the design of targeted drugs .
notably , because ra is a systemic autoimmune disease characterized by chronic inflammation of multiple joints and proinflammatory cytokines such as tumour necrosis factor ( tnf)- , interleukin ( il)-1 , il-6 , or il-17 may be reconsidered as the etiology for ra and further leads to the development of targeted therapies [ 3 , 911 ] .
indeed , tnf- inhibitors , anti - tnf antibodies , a soluble tnf receptor fusion protein , and il-1 receptor antagonist have been attempted for the treatment of ra , but their side effects such as serious infections and inducible malignant tumors still remain not to be resolved . moreover , about 25~30% of ra patients treated with tnf antagonists do not display any significant clinical improvement after initiation of therapy .
patients will display feature of secondary resistance to the delivered drug within the first 5 years of therapy .
all these findings raise a question that it is still incompletely understood on the relationship between inflammation inhibitions and ra therapy . in mammalian cells , nuclear factor-b ( nf-b )
it includes subunits as nf-b1 ( p50/p105 ) , nf-b2 ( p52/p100 ) , rela ( p65 ) , c - rel , and relb and regulates immune and inflammatory responses , cellular proliferation , or cell death [ 12 , 13 ] .
nf-b is constitutively activated in many autoimmune diseases , including ra [ 14 , 15 ] .
for example , flss release cytokines , chemokines , and growth factors to sustain inflammation and produce enzymes that degrade the organized extracellular matrix , destroying cartilage and bone [ 16 , 17 ] .
moreover , nf-b activation facilitates synovial hyperplasia by promoting proliferation and inhibiting apoptosis of flss .
thus , nf-b seems to be a positive regulator of cell growth and inhibitor of cell apoptosis in flss .
however , some studies have suggested that nf-b may also function in a proapoptotic fashion .
for example , induction of apoptosis in human embryonic kidney cell line ( 293 cells ) after serum withdrawal requires nf-b activation .
nf-b activation may promote apoptosis in neural cells   and t cell hybridomas , and high levels of c - rel induce apoptosis in avian embryos and in bone marrow cells in vitro .
thus , the functions of nf-b in apoptosis may be distinct by different cell types .
at least , all the paradox indicates that inflammations mediated by nf-b may not always be beneficial for cell survival .
ectopic elevations of inflammations may afford us new insights into the clinical ra therapy and explain why sometimes anti - inflammation drugs are not so efficacious . in this study
, we proposed to determine whether enhanced inflammations may lead to cell apoptosis in flss .
thus , we screened and identified the inflammation alternations by 4-hydroxynonenal ( 4-hne ) treatment in mh7a cells , a human rheumatoid arthritis synovial cell line
. 4-hne is an aldehyde product of membrane lipid peroxidation , generated through peroxidation of omega 6-polyunsaturated fatty acids [ 24 , 25 ] .
our results showed that , by 4-hne treatment , flss develop strong inflammation reactions , such as elevated il1- , il-6 , and tnf- transcriptions and cox-2 expressions .
of note , 4-hne induces nf-b activation , indicated by rela ( p65 ) nuclear translocations and subsequent cell apoptosis .
the present findings may help to understand the molecular network of inflammations and apoptosis in flss and offer novel insights into the clinical improving of ra therapy .
dulbecco 's modified essential medium ( dmem ) , fetal bovine serum ( fbs ) , and bay11 - 7082 ( nf-b inhibitor ) were purchased from gibco invitrogen ( carlsbad , ca , usa ) .
the millipore luminex kits for cell signaling multiplex assay were from millipore ( billerica , ma , usa ) ( # 48 - 611mag and 48 - 680mag ) .
the hoechst kit for cell death detection was from beyotime biotechnology ( haimen , jiangsu , china ) .
the following antibodies , anti - cox-2 , anti - lamin a / c , anti - beta actin , and anticleaved caspase 3 were from santa cruz biotechnology ( santa cruz , ca , usa ) . the nf-b ( p65 ) and gapdh antibodies were from cell signaling technology ( danvers , ma , usa ) .
a widely used mh7a human rheumatoid arthritis synovial cell was chosen as in vitro experiment system , which was obtained from shanghai institute of cell biology ( introduced from american type culture collection ) . in our experiments ,
mh7a cells were plated in 6-well plates at 1.0  10  cells / ml . the cells were incubated in dulbecco 's modified essential medium ( dmem ) containing 10% fetal bovine serum ( fbs ) plus antibiotics for 24  h in 5% co2 at 37c . for 4-hne and following bay11 - 7082 treatment ,
the final low and high concentrations ( 5  m and 50  m ) of 4-hne and 10  m bay11 - 7082 were applied to these cells and then incubated from 0 to 12  h. no additives were used as internal controls . after culturing , the cells were harvested for subsequent examinations . to screen the targeted intracellular signaling of 4-hne on mh7a cells , cell signaling multiplex assay was carried out by millipore luminex kits . briefly ,
the cell samples were loaded to 96 wells and coated with respective primary antibody mixtures .
after washing , samples were detected in luminex multiplexing instruments ( millipore , billerica , ma ) .
rna was subjected to reverse transcription with reverse transcriptase as the manufacturer 's instructions ( fermentas ) .
quantitative real - time pcr was performed using the bio - rad iq5 system , and the relative gene expression was normalized to internal control as beta actin .
primer sequences for sybr green probes of target genes are as follows , il-1 
 : ctggtgtgtgacgttcccatta and ccgacagcacgaggcttt ; il-6 : ttccatccagttgccttcttg and ttggg agtggtatcctctgtga ; tnf- 
 : catcttctcaaaattcgagtgaca and tgg gagtagacaaggtacaaccc .
mh7a cell samples were lysed in lysis buffer , and insoluble material was removed by centrifugation at 12,000  g for 20  min at 4c .
final protein concentrations were determined using the bradford protein assay ( bio - rad laboratories ) .
electrophoresis was performed using sds - page , and blots were transferred to nitrocellulose membranes .
resulting films were scanned , and optical densities were quantified using imagej . to detect the nf-b nuclear translocations ,
mh7a cells were harvested and isolated into cytosol and nuclear parts , according to instructions of nuclear / cytosol fractionation kit ( biovision ) . as for the nf-b activity detections ,
plasmids encoding nf-b - responsive firefly luciferase and renilla luciferase , which is an internal control for normalizing transfection , were transfected to mh7a cells using lipofectamine 2000 .
after transfection for 0 , 3 , or 12  h , cells were washed with phosphate - buffered saline , serum - starved in opti - mem overnight , and then assayed for luciferase activity using the dual - luciferase reporter assay kit ( promega ) . for the preparation of cell death detection by hoechst staining , mh7a cells were plated with 1.0  10  cells / ml in 6-well plates . after pharmacological manipulations , cells were directly stained with hoechst kit from beyotime .
the cell counting was carried out through the use of national institutes of health software imagej , which is available from http://rsbweb.nih.gov/. data represent the mean and standard error of the mean ( sem ) .
student 's t - test was performed for all statistical significance analysis using graphpad prism software .
to screen the effects of lipid peroxidation on synoviocytes , we examined the intracellular pathways by millipore luminex kits after 4-hne treatment .
it is noted that , after 4-hne treatment , inflammation relative pathways , such as nf-b and stats , were significantly upregulated ( figure 1 ) , while the cell growth or survival relative pathways , such as insulin receptor ( ir ) , akt , mtor , and gsk pathways , were downregulated ( figure 1 ) . these results are consistent with the previous findings , indicating that lipid peroxidation is able to induce cell stress to promote inflammations and inhibit cell growth . as for the ectopic upregulation of inflammations by 4-hne treatment , we determine to identify the subsequent inflammation alternations and cell viability in mh7a synoviocytes . to further confirm the inflammation inductions by 4-hne treatment , we next examined the expressions of canonical inflammation factors , including il1- , il-6 , and tnf- .
the results of quantitative real - time pcr showed that under low concentrations of 4-hne ( 5  m ) , the mrna levels of il1- , il-6 , and tnf- were increased gradually with the increasing time up to 12  h , and the maximum folds increased by 6.79 , 6.19 , and 4.28 compared to the control , respectively ( figures 2(a ) , 2(b ) , and 2(c ) ) .
interestingly , when the concentration of 4-hne increased to 50  m , the mrna levels of il1- , il-6 , and tnf- in rheumatoid arthritis synovial cells increased gradually with the increasing time up to 1  h and then decreased ( figures 2(d ) , 2(e ) , and 2(f ) ) .
the shorter peak time of mrna increasing may indicate that inflammations are enhanced by high lipid peroxidation treatment . and
the overwhelmed lipid peroxidation may terribly affect normal cell functions and lead to a subsequent reduction of inflammation gene transcriptions .
nevertheless , our results support the notion that lipid peroxidation may indeed induce inflammation reactions in synoviocytes . to further confirm the inflammation alternations by lipid peroxidation
, we next examined the protein level of cox-2 in 4-hne - treated mh7a cells .
cox-2 is an inducible isoform of prostaglandin h synthase , which mediates prostaglandin synthesis during inflammations .
we found that , under 5  m 4-hne conditions , cox-2 protein levels were increased gradually with the time up to 12  h , with the highest value being about 2.86-folds that of the control ( figures 3(a ) and 3(b ) ) .
this indicates that lipid peroxidations may induce cox-2 expressions in a time - dependent manner in mh7a synovial cells .
when the 4-hne concentration came to 50  m , the protein levels of cox-2 were increased gradually with the increasing time up to 1  h and then decreased ( figures 3(c ) and 3(d ) ) .
these alternations are consistent with the changes of inflammation gene transcriptions , suggesting that lipid peroxidations could enhance inflammation reactions in rheumatoid arthritis synoviocytes .
inflammation is controlled by multiple signaling pathways in mammalian cells , and nf-b plays a pivotal role in the regulation of inflammatory responses . by millipore luminex kits
, we have identified that nf-b pathway is induced by 4-hne . to confirm this finding
, we further investigated the nf-b activations by 4-hne treatment . upon activation of nf-b by stimulators , nf-b subunits ( especially for p65 ) translocate into the nucleus to activate transcription of genes involved in cell inflammations or proliferations .
accordingly , we evaluated nf-b activity by detecting the nuclear localization of the p65 subunit .
subcellular fractionation of low 4-hne - treated mh7a cells showed that p65 localization was enhanced in the nucleus compared to controls ( figure 4(a ) ) .
an nf-b - re - luc reporter assay also demonstrated that nf-b activity was 10.1-fold greater after 12  h 4-hne treatment compared with the level in controls ( figure 4(b ) ) . under high 4-hne concentrations , nf-b translocation into nucleus
was increased as early as 3  h but decreased at 12  h ( figure 4(c ) ) .
the nf-b activity assay also exhibited that nf-b activity was increased in 3  h and decreased in 12  h , respectively .
thus , these results indicate that nf-b activity could be induced by lipid peroxidations , and overwhelmed oxidative stress may even impair the nf-b activation conversely .
lipid peroxidation has been proved to induce nf-b pathway and inflammatory responses in mh7a cells ; we wonder how these inflammations affect cellular functions , especially on cell viability . to examine whether 4-hne treatment may induce the cell apoptosis in mh7a cells , we applied hoechst staining to mh7a cells treated by 4-hne .
quantitative results showed that 5  m 4-hne could induce dramatic cell apoptosis in mh7a cells , with nearly 87.8% cell apoptosis in the time of 12 hours ( figures 5(a ) and 5(b ) ) .
comparably , high concentration 4-hne induces faster cell apoptosis than that in low 4-hne group ( figures 5(a ) and 5(c ) ) .
we also examined cleaved caspase 3 , a well - known apoptotic marker to confirm the cell apoptosis by 4-hne .
results showed that the cleaved caspase 3 increased as 4-hne treatment in a time- and dose - dependent manner .
moreover , our findings also indicated that the cleaved caspase 3 increased and total caspase 3 decreased as 4-hne treatment in a time- and dose - dependent manner ( figures 5(d ) and 5(e ) ) .
it is noted that the protein level of cleaved caspase 3 seemed to decrease in 50  m 4-hne for 12  h , which could be explained as the solemn impairment of cell structures and functions for overwhelming 4-hne treatment . based on the above results
since nf-b is a master regulator of inflammatory responses , we would like to test whether inhibition of nf-b activation could reduce inflammations and subsequent cell apoptosis in mh7a cells .
firstly , we examined the inflammatory responses by bay11 - 7082 ( nf-b inhibitor ) treatment in mh7a cells under the condition of 4-hne .
the mrna levels of il1- , a marker of inflammatory response , was lower in the presence of bay11 - 7082 compared to that of 4-hne alone group , either in low or high concentration of 4-hne group ( figures 6(a ) and 6(b ) ) .
moreover , the induction of cox-2 was also reduced by bay11 - 7082 treatment ( figures 6(c ) and 6(d ) ) .
these findings indicate that the inhibition of nf-b signaling attenuates inflammatory responses in rheumatoid arthritis synoviocytes . since inhibition of nf-b activation could reduce inflammations in mh7a cells , we further investigate whether the nf-b inhibitor could reduce 4-hne mediated apoptosis in mh7a cells .
quantitative results showed that in the presence of bay11 - 7082 , the percentage of cell apoptosis reduced compared to the counterparts without bay11 - 7082 under 5  m
while in 50  m 4-hne groups , the protective effect of bay11 - 7082 was weakened , especially in 6 and 12  h groups ( figure 7(b ) ) .
this may be due to the solemn impairment of cell structures and functions by overwhelming lipid peroxidations .
nevertheless , these data at least verify that inhibition of nf-b and inflammations could partly reduce the 4-hne mediated cell apoptosis in rheumatoid arthritis synoviocytes .
in the present study , we reveal a novel mechanism to clarify the role of inflammations on ra development .
we demonstrate that products of lipid peroxidations , 4-hne , could induce synovial intrinsic inflammations by activating nf-b pathways and lead to cell apoptosis .
pharmacological inhibition of nf-b activation may reduce the 4-hne mediated inflammations and subsequent cell apoptosis ( figure 8) .
our work uncovers novel relationships between inflammations and ra development and offers new strategy to ra clinical therapy .
rheumatoid arthritis ( ra ) is a chronic inflammatory disease of synovium that can lead to severe joint damage .
the central pathogenesis of ra is a proliferation of fibroblast - like synoviocytes ( flss ) in response to stimulators . during the process of flss proliferation ,
previous studies focus on drugs to inhibit inflammations , but they sometimes do not display significant clinical improvement after initiation of therapy , as well as bringing side effects such as serious infections and inducible malignant tumors .
now , our results could partly answer the question that why sometimes anti - inflammation drugs do not perform well in ra therapy .
at least , the inhibitory effect of anti - inflammation drugs may partly block the inflammation - mediated cell apoptosis in rheumatoid arthritis synoviocytes . therefore
, appropriate drug compatibility should be important to balance the effect of inflammations on ra development and flss apoptosis .
since inflammation balance is important for the survival of flss , the precise regulation of synovial inflammations must be well achieved .
it has been long appreciated that nf-b is a significant transcription factor that functions in immune and inflammatory responses .
for example , nf-b plays a pivotal role in myocardial ischemia - reperfusion injury and induces many proinflammatory cytokines and chemokines .
moreover , nf-b is also thought to act as a redox sensitive transcription factor , that has been proposed to be the sensor for oxidative stress .
reactive oxygen species ( ros ) can activate nf-b directly , and ros is also involved in nf-b activation by other stimuli such as tnf . in the present study ,
we demonstrate that products of lipid peroxidation , 4-hne , may induce cell death in rheumatoid arthritis synovial cells by activating nf-b pathways .
our results confirmed that ros and its relative products , that is 4-hne , are enough to induce inflammation reactions via nf-b . considering that ectopic activation of nf-b induces synovial apoptosis
, it may afford us some new strategy to design antirheumatic drugs targeted at nf-b .
besides the ros itself , the products of lipid peroxidation may also cause the formation of reactive aldehydes and lead to inflammations .
these lipid peroxidations have longer biological half - lives than free radicals and can diffuse from their site of formation to reach distant targets and cause cellular damage .
4-hne is an  , -unsaturated hydroxyalkenal that is produced by lipid peroxidation in cells [ 32 , 33 ] .
it is found throughout animal tissue and in higher quantities during oxidative stress for the increase in the lipid peroxidation chain reaction and for the increase in stress events [ 34 , 35 ] .
however , the  normal  level of lipid peroxidation is limited to a low level , and even the modest upregulation could alter multiple intracellular pathways .
thus , we propose that lower levels of intracellular 4-hne are beneficial to cells , but higher levels can cause a toxic response in the cell and may lead to cell death . in this study , we applied different doses of 4-hne to treat mh7a synovial cells for different time points .
interestingly , we note that even a short overwhelmed treatment of 4-hne exhibits pretty effects on synovial apoptosis .
notably , the effect of low concentration 4-hne could be partly restored by nf-b inhibitors , while high concentration 4-hne could not .
this suggests that at least the effects of lipid peroxidation to synovial apoptosis might be partly dependent on nf-b mediated inflammation reactions .
in conclusion , the results of this study support the notion that lipid peroxidation - mediated inflammation promotes cell apoptosis through activation of nf-b pathway in rheumatoid arthritis synovial cells .
our results imply that blocking nf-b activation may be partly beneficial for human ra therapy .
 rheumatoid arthritis ( ra ) is a systemic autoimmune disease characterized by chronic inflammation of multiple joints . 
 the central pathogenesis of ra is the proliferation of synovial fibroblasts in response to inflammatory cytokines . 
 however , some of the targeted therapies for inflammation reactions do not display significant clinical improvement after initiation of therapy . 
 thus , the relationship between inflammatory responses and ra therapy is still incompletely understood . in the present study 
 , we proposed to determine whether enhanced inflammations may lead to cell apoptosis in rheumatoid arthritis synoviocytes . 
 our results indicated that products of lipid peroxidations , 4-hne , may induce synovial intrinsic inflammations by activating nf-b pathways and it may lead to cell apoptosis . 
 pharmacological inhibition of nf-b activation may reduce the 4-hne mediated inflammation responses and subsequent cell apoptosis . 
 our results may help to clarify the role of inflammations on ra development and imply that blocking nf-b activation may be partly beneficial for human ra therapy . 
 these findings might provide a mechanism - based rationale for developing new strategy to ra clinical therapy . 