oversecretion of proinflammatory factors from osteoblasts plays vital roles in the progress of osteoarthritis [ 1 , 2 ] , and high levels of proinflammatory factors in bones and joints induce pain , cartilage loss , and even joint dysfunction [ 3 , 4 ] . therefore , reducing the release of proinflammatory factors from osteoblasts is an effective therapy for oa . traditional anti - inflammatory drugs , including steroidal anti - inflammatory drugs and nonsteroid anti - inflammatory drugs ( nsaid ) , are effective in alleviating the symptoms of oa [ 5 , 6 ] . however , long - term use of these drugs brings about many side effects , such as gastritis , gastric ulcer , and/or immune suppression [ 5 , 7 ] . therefore , a novel anti - inflammatory drug with mild side effects is needed urgently in the treatment of oa . cannabinoid 9-tetrahydrocannabinol ( thc ) is a main bioactive component from marijuana , and thc is effective in treating oa ; however , the mechanism is still not clear . activation of cb1 receptor is associated predominantly with a dampening down of neuronal excitability , whereas activation of cb2 receptors is related to reducing immune cell function , including decreases of proinflammatory factors release . therefore , we aimed at cb2 receptor , not cb1 , as one of the research targets in this study . cofilin is a cytoskeleton protein family , including nonmuscle cofilin ( cofilin-1 ) , muscle cofilin ( cofilin-2 ) , and actin depolymerizing factor ( adf ) . moreover , cofilin-1 is associated with many inflammatory conditions , such as oa and inflammatory pain [ 13 , 14 ] , and some investigations indicated that overexpression of cofilin-1 can decrease glucocorticoid receptor expression and nf-b activity [ 15 , 16 ] . however , whether cofilin-1 is involved in the therapeutic effects of thc in treating oa is still unclear . mg-63 cell is an osteosarcoma cell - line , used widely to mimic osteoblasts in vitro . lipopolysaccharide ( lps ) is a component of the outer membrane of gram - negative bacteria , which has been used mainly as a proinflammatory substance in several investigations . and mg-63 cells exposed to lps can be used as an inflammatory model of oa in vitro . in this study , we hypothesized that cannabinoid cb2 receptor is involved in thc - induced anti - inflammation in lps - stimulated mg-63 cells , and the thc - induced anti - inflammation may be mediated by cofilin-1 protein . mg-63 , an osteogenic human osteosarcoma cell - line , was obtained from the america type culture collection ( atcc ) . cells were cultured in minimum essential medium eagle with earl 's salts ( mem ) medium ( hyclone , usa ) containing 10% fetal bovine serum ( hyclone , usa ) , 100  u / ml penicillin , and 0.1  mg / ml streptomycin in a humidified atmosphere containing 5% co2 and 95% air at 37c . thc , lps , and doxycycline were purchased from sigma - aldrich ( st . louis , mo , usa ) . cb2 antagonist am630 and cb1 antagonist am251 were obtained from tocris bioscience ( uk ) . anti - cb2 antibody and anti - cofilin-1 antibody were purchased from the abcam ( cambridge , uk ) . the mg-63 cells were divided into five groups , including control , lps , thc + lps , am630 + thc + lps , and am630 alone groups . after the treatments , immunocytochemistry staining and western blot were used to investigate the expression of cb2 receptor in the cells ( figure 1(a ) ) . then , the cells were assigned into seven groups , including control , lps , thc + lps , am630 + thc + lps , am630 alone , am251 + thc + lps , and am251 alone groups . after the treatments , inflammatory factors levels in the supernatants were evaluated by enzyme - linked immunosorbent assay ( elisa ) kits ( figure 1(b ) ) . to further determine the role of cb2 in the thc - induced anti - inflammatory effects in mg-63 cells , the cells were divided into five groups , including control , lps , thc + lps , cb2-sirna + thc + lps , and scrambled- ( sc- ) sirna + thc + lps groups , after the treatments as shown in this figure , and western blot and elisa kits were used to evaluate nf-b expression and inflammatory factors release , respectively ( figure 1(c ) ) . then the mg-63 cells were divided into five groups , including control , lps , thc + lps , doxycycline ( dox ) + thc + lps , and dox alone groups , after the treatments , and western blot and elisa kits were used to evaluate cofilin-1 expression and inflammatory factors release , respectively ( figure 1(d ) ) . the release of tnf- , il-1 , il-6 , and il-8 from the lps - stimulated mg-63 cells was determined by the corresponding elisa kit ( r & d systems , minneapolis , mn , usa ) according to the instructions . the mg-63 cells were seeded in a 24-well plate at a density of 5  10 cells / well . after the incubation for 24  h , the supernatants were collected and centrifuged , and then the cell - free supernatants were used for elisa assay . the mg-63 cells were seeded into five confocal microscopy special dishes at a density of 2  10 cells / dish . after the treatments , the mg-63 cells were washed three times with phosphate buffer solution ( pbs ) and then fixed with 4% paraformaldehyde solution for 30  min and blocked with 50  mg bovine serum albumin ( bsa)/ml in pbs for 30  min . cells were incubated with primary anti - cb2 antibody ( 1  :  50 ) overnight at 4c and then washed three times with pbs , before incubation with cy3-labeled secondary antibody ( 1  :  200 , beyotime , china ) for 1  h at room temperature . the 4,6-diamidino-2-phenylindole ( dapi ) staining solution ( 200  l ) was added into each dish for 5  min , and then the dishes were washed three times with pbs . cb2 expression was observed by a laser scanning confocal microscope ( fv10i , olympus , japan ) ( excitation = 550  nm , emission = 570  nm ) . after the treatments , the mg-63 cells were washed twice with ice - cold pbs ( ph 7.2 ) and harvested with ice - cold pbs and then centrifuged at 14000  g for 5  min at 4c . nuclear and cytosolic extracts were prepared using a nuclear and cytoplasmic protein extraction kit ( beyotime , china ) according to the manufacturer 's instructions . the samples were fractionated on 10% sds - polyacrylamide gel electrophoresis ( sds - page ) and transferred to polyvinylidene fluoride membranes ( chemicon international , millipore , billerica , ma , usa ) . the membranes were blocked for 1  h at room temperature with 5% nonfat milk in pbs - tween 20 ( 0.1% ) and then incubated with different primary antibodies ( cb2 , 1  :  200 ; nf-b , 1  :  2000 ; cofilin , 1  :  2000 ) . the membrane was incubated with electrochemiluminescence reagent for 10  min and exposed to x - ray film . the signals were quantified by densitometry using a western blotting detection system ( alpha innotech , usa ) . mg-63 cells were transfected with specific cb2 targeting small interfering rna ( sirna ) designed to knockdown cb2 protein expression ( dharmacon , usa ) . briefly , mg-63 cells ( 70% confluent ) were transfected with 100  pmol cb2-sirna or scrambled- ( sc- ) sirna using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's protocol . after the transfection for 24  h , the results of this study were expressed as means  sd , which were obtained from at least three independent experiments for each condition . the statistical significance of the results was evaluated with one - way variance analysis ( one - way anova ) , followed by tukey 's multiple comparison test . the mg-63 cells were exposed to different concentrations of lps for 24  h ( figure 2(a ) ) , causing a dose - dependent release of il-6 from the cells . il-6 is a proinflammatory factor , which was evaluated as an index reflecting the inflammation level of the lps - treated mg-36 cells . the presence of 10  ng / ml lps for 24  h increased the il-6 level to 714.2  55.4  pg / ml , compared with 38.5  13.2  pg / ml of the control ( p < 0.05 ) in the supernatants . coadministration of 0.5 , 5 , and 50  m thc significantly reduced the il-6 release from the lps - stimulated cells ( figure 2(b ) ) , suggesting that thc induced an anti - inflammation . immunocytochemistry and western blot were used to determine the expression of cb2 protein in mg63 cells . we observed that lps alone did not induce a marked change of cb2 protein expression ( figures 3(a)-3(b ) ) , and thc ( 5  m ) upregulated cb2 protein expression . however , coadministration of cb2 antagonist am630 ( 10  m ) significantly inhibited the thc - induced upregulation of cb2 expression and did not induce a marked change of cb2 expression alone . incubation with lps ( 10  ng / ml ) for 24  h sharply increased the concentrations of tnf- , il-1 , il-6 , and il-8 in the supernatants ( p < 0.05 ) and the upregulation of nf-b expression in mg-63 cells , and coadministration of thc ( 5  m ) significantly reduced the release of the proinflammatory factors ( figures 4(a)4(d ) ) and the upregulation of nf-b ( figures 5(a)-5(b ) ) . however , cb2 antagonist am630 , not cb1 antagonist am251 , inhibited the thc - induced reduction of release of proinflammatory factors and nf-b expression , indicating that the thc - induced anti - inflammatory effects may be mediated by cb2 receptor , not by cb1 . to further determine whether the thc - induced anti - inflammation was mediated by cb2 receptor in mg-63 cells , we used cb2-sirna to knockdown the cb2 protein expression in mg-63 cells . we found that cb2-sirna was effective in reducing the expression of cb2 ( figure 6(a ) ) . in addition , cb2-sirna reversed the thc - induced inhibition of nf-b expression ( figure 6(b ) ) and proinflammatory factors release ( figures 6(c)6(f ) ) , including tnf- , il-1 , il-6 , and il-8 ( p < 0.05 ) . incubation with 10  ng / ml lps for 24  h markedly increased the cofilin-1 expression in mg-63 cells ( p < 0.05 ) compared with the cells cultured in drug - free medium , and coadministration of 5  m thc inhibited the cofilin-1 expression significantly ( figure 6(a ) ) . cb2 antagonist am630 partially reversed the thc - induced inhibition of cofilin-1 expression . in order to further determine the role of cofilin-1 in thc - induced anti - inflammation in the mg-63 cells , we used doxycycline , which can upregulate the expression of cofilin-1 significantly in vitro [ 20 , 21 ] . we observed that 0.1  g / ml doxycycline markedly increased the expression of cofilin-1 in mg-63 cells ( figure 6(b ) ) . and coadministration of 0.1  g / ml doxycycline partially reversed thc - induced decrease of il-6 and tnf- release ( figures 6(c)-6(d ) ) . these findings indicated that cofilin-1 was involved in the thc - induced anti - inflammation via cb2 in the mg-63 cells exposed to lps . in this study , we found that the presence of 5  m thc attenuated the inflammation in mg-63 cells exposed to 10  ng / ml lps . meanwhile thc increased cb2 protein expression and inhibited the upregulation of nf-b and cofilin-1 expression in the mg-63 cells . however , administration of cb2 antagonist am630 or cb2-sirna , not cb1 antagonist am251 , partially reversed the thc - induced anti - inflammatory effects . moreover , upregulation of cofilin-1 protein partially abolished the thc - induced anti - inflammation ( figure 7 ) . these findings indicated that cb2 receptor is involved in the thc - induced anti - inflammation in the mg-63 cells exposed to lps , and the anti - inflammation may be mediated by cofilin-1 protein . oa is one of the most common causes of disability in elder adults , especially in the population aged over 65 years . oversecretion of proinflammatory cytokines , including tnf- , il-6 , and il-1 , contributes to the severity and the progression of oa . high levels of proinflammatory cytokines in bones and joints can damage articular cartilage by inducing chondrocyte apoptosis and proteoglycan depletion . therefore , inhibiting the secretion of proinflammatory cytokines is vital to prevent the progression of cartilage degeneration at the initial stage of oa . at present , corticosteroids and nsaid are used widely to attenuate the inflammation of oa [ 23 , 24 ] . as these drugs just relieve the symptoms of oa rather than cure the disease , side effects , including vomiting , gastritis , and immune suppression , emerged with repeated and long - term use [ 5 , 25 ] . thus , a drug that can treat oa with slight and less side effects is needed urgently . thc is main cannabinoid compounds from marijuana , which has a number of therapeutic applications , including anti - inflammation and analgesic effects . however , thc has rarely been well studied in treating oa and its therapeutic mechanism is still not clear . in most cases , thc is used as a painkiller , which relieves pain via activating cannabinoid and opioid receptors expressed in the brain . therefore , researchers paid more attention to the cns system rather than the peripheral tissue during investigating the mechanism of thc in anti - inflammation . in this study , we found that thc attenuated lps - induced proinflammatory factors release from the mg-63 cells , including tnf- , il-1 , il-6 , and il-8 , and thc reduced the upregulation of nf-b expression in the mg-63 cells , indicating that the thc - induced anti - inflammatory effects may be associated with its anti - inflammation . moreover , selective cb2 receptor antagonist am630 or cb2-sirna , not cb1 antagonist am251 , significantly reversed the thc - induced anti - inflammatory effects and the inhibition of nf-b expression , suggesting that the thc - induced anti - inflammation may be mediated by cb2 receptor in mg-63 cells . as one of the key molecules regulating the release of various inflammatory factors , the expression level of nf-b reflects the inflammation degree in cells . nf-b locates in the nucleus , and cb2 is mainly expressed in the cell membrane and cytoplasm [ 28 , 29 ] ; thus we infer that thc may activate cb2 receptors expressed in the cell membrane and cytoplasm of mg-63 cells and then inhibit nf-b expression to reduce the inflammatory factors release from the mg-63 cells exposed to lps . similarly , gondim et al . reported that cb2 is involved in electroacupuncture- ( ea- ) induced anti - inflammatory effects in a rat model of arthritis . and zarruk et al . reported that the activation of microglial cb2 receptor brings about neuroprotection against brain ischemic injury by decreasing the proinflammatory cytokines release in mice . although thc is a cannabinoid ligand for both cb1 and cb2 receptors , newton and klein described that thc downregulates serum ige levels through cb2 receptor rather than cb1 receptor in an ige induction model in mice . these findings supported that activation of cb2 receptor may be involved in thc - induced anti - inflammation in mg-63 cells . cofilin is a cytoskeleton protein family , which is known to promote actin depolymerization and maintain the stability of cell structure . a study indicated that overexpression of cofilin can induce glucocorticoid resistance , and another study mentioned that jwh015 , a selective cb2 agonist , modulated the activity of cofilin . cofilin is also an important signaling molecule for the cytoskeletal and migratory properties of trabecular meshwork cells . the cofilin protein family consists of three isoforms , including cofilin-1 , cofilin-2 , and adf . cofilin-1 and adf are mainly expressed in non - muscle tissue , and cofilin-1 predominates . chiang et al . reported that cofilin-1 is involved in isoflurane- ( an inhaled anesthetic ) induced anti - inflammatory effect ; besides cb2 could be activated by isoflurane , suggesting that cofilin-1 may be involved in the modulation of inflammation , which might be related to cb2 receptor . therefore , we investigated cofilin-1 in thc - induced anti - inflammatory effects in this study . we observed that cb2 antagonist am630 reversed the thc - induced modulation of cofilin-1 expression in the lps - stimulated mg-63 cells , and overexpression of cofilin-1 abolished the thc - induced anti - inflammation in the cells , indicating that cofilin-1 may mediate the thc - induced anti - inflammatory effects via cb2 receptor . moreover , rom et al . reported that cb2 selective agonists gp1a and am1241 increase the phosphorylation of cofilin in brain tissue , causing protection of blood - brain barrier against inflammatory leukocyte . therefore , we infer that thc - induced anti - inflammation may be related to the phosphorylation of cofilin in mg-63 cells exposed to lps . first , our study is in vitro ; thus the results of this investigation should be further identified in in vivo study and clinical trials . second , the mg-63 cells used here are an osteosarcoma cell - line , not primary osteoblasts , so the results of this study should be accepted cautiously . third , we just upregulated the cofilin-1 expression in this study ; how thc works is still unknown when cofilin-1 is downregulated . our study indicates that cannabinoid cb2 receptor is involved in thc - induced anti - inflammation in mg-63 cells exposed to lps , and the anti - inflammatory effects may be mediated by cofilin-1 protein .
cannabinoid 9-tetrahydrocannabinol ( thc ) is effective in treating osteoarthritis ( oa ) , and the mechanism , however , is still elusive . 
 activation of cannabinoid receptor cb2 reduces inflammation ; whether the activation cb2 is involved in thc - induced therapeutic action for oa is still unknown . 
 cofilin-1 is a cytoskeleton protein , participating in the inflammation of oa . in this study , 
 mg-63 cells , an osteosarcoma cell - line , were exposed to lipopolysaccharide ( lps ) to mimic the inflammation of oa . 
 we hypothesized that the activation of cb2 is involved in thc - induced anti - inflammation in the mg-63 cells exposed to lps , and the anti - inflammation is mediated by cofilin-1 . 
 we found that thc suppressed the release of proinflammatory factors , including tumor necrosis factor  ( tnf- ) , interleukin- ( il- ) 1 , il-6 , and il-8 , decreased nuclear factor-b 
 ( nf-b ) expression , and inhibited the upregulation of cofilin-1 protein in the lps - stimulated mg-63 cells . however , administration of cb2 receptor antagonist or the cb2-sirna , not cb1 antagonist am251 , partially abolished the thc - induced anti - inflammatory effects above . 
 in addition , overexpression of cofilin-1 significantly reversed the thc - induced anti - inflammatory effects in mg-63 cells . 
 these results suggested that cb2 is involved in the thc - induced anti - inflammation in lps - stimulated mg-63 cells , and the anti - inflammation may be mediated by cofilin-1 .