Study Of Effects And Mechanisms Of Salicylate Sodium On MCP-1 Production In TNF-α-stimulated Adipocytes

Obesity leads to chronic,low-intensity inflammation called metaflammation(also known as "cold inflammation").Many obesity-related metabolic diseases,such as insulin resistance,non-alcoholic fatty liver disease,and type 2 diabetes mellitus,are closely associated with the occurrence and development of "cold inflammation",rather than "hot inflammation" characterized by "redness,swelling,heat,and pain".Adipose tissue inflammation plays an important role in metaflammation.Although moderate inflammation is beneficial for the expansion and remodeling of adipose tissue,overnutrition will cause adipocytes hyperplasia(mostly subcutaneous)and hypertrophy(mostly visceral).Lipid overload can cause hypertrophic adipocytes to suffer from hypoxia,leading to stress response and even death,which can initiate "cold inflammation" in adipose tissue.An important part of this process is that adipocytes release large amounts of MCP-1 to recruit circulating monocytes/macrophages to adipose tissue.Monocytes/macrophages that migrate to adipose tissue can secrete TNF-α to stimulate adipocytes to produce more MCP-1,thus forming the vicious cycle,which may be the reason why metaflammation is difficult to subside naturally.Therefore,inhibiting excessive production of MCP-1 in TNF-α-activated adipocytes may be an effective strategy to break this vicious cycle and prevent the occurrence and development of obesity-related metabolic diseases.Salicylic acid drugs are classical non-steroidal anti-inflammatory drugs,widely used in clinical treatment of "hot inflammation"-related diseases.In terms of its mechanism,these drugs can not only decrease the production of inflammatory mediator PGE2 by non-selectively inhibiting COX,but also prevent NF-κB activation by directly inhibiting IKKβ activity.In addition to the application of "hot inflammation",recent clinical studies have reported that salicylic acid drugs(such as aspirin and salsalate)can also improve"cold inflammation" related diseases,such as insulin resistance and type 2 diabetes mellitus.Although this effect is also thought to be related to inhibiting IKKβ activity,whether they work through the same target on "hot" and "cold" inflammation has not been confirmed.Based on this,we carried out the following research.In a high fat diet-induced obese mouse model,fluorescent microspheres which could be phagocytized by monocytes were injected intravenously to trace cell migration.The results showed that salicylate sodium could significantly prevent circulating monocytes from migrating to adipose tissue in obese mice.Since the chemotaxis of monocytes are mainly recruited by MCP-1 at the chemotaxis destination,we investigated the effect and molecular mechanism of salicylate sodium on MCP-1 production in TNF-α-activated adipocytes.In three types of adipocyte models(3T3-L1 pre-adipocytes,mature 3T3-L1 adipocytes,and mouse primary adipocytes),TNF-α(40 ng/mL)could significantly stimulate adipocytes to produce MCP-1,and salicylate sodium suppressed MCP-1 production in three types of adipocytes.Among three types of adipocytes,3T3-L1 pre-adipocytes exhibited higher reactivity in response to TNF-α.Moreover,this cell line possesses proliferation capacity.Thus,3T3-L1 pre-adipocytes were used for most of the subsequent experiments.The results showed that salicylate sodium could significantly decrease MCP-1 mRNA level in TNF-α-activated adipocytes.without affecting the mRNA stability.Based on the fact that salicylate sodium can inhibit IKKβand activate AMPK,we first used gene silencing technology to specifically knock down IKKβ in adipocytes.The results showed that the inhibitory effect of salicylate sodium on MCP-1 production was not affected in IKKβ-deficient 3T3-L1 pre-adipocytes.And AMPK inhibitor also didn’t affect the effect of salicylate sodium.Indeed,IKKβ siRNA,multiple NF-κB inhibitors with different mechanisms,or AMPK activators all failed to suppress MCP-1 production in TNF-α-activated adipocytes.In contrast,the inhibitors of anyone of MAPKs(JNK,ERK and p38)could do.And salicylate sodium indeed decreased the phosphorylation level of ERK and p3 8,and increased the expression of their phosphatase MKP-1.Since MKP-1 expression was regulated by cAMP/PKA signaling,the effect of salicylate sodium on this signaling pathway was investigated.The results showed that salicylate sodium increased intracellular cAMP level and activated its effector protein PKA,and cAMP antagonists RP-cAMPS and PKA inhibitor H-89 both could inhibit the effect of salicylate sodium.Subsequently,we investigated the effect of salicylate sodium on the hydrolase of cAMP,PDE.By using SPR technology,we found that salicylate sodium can directly bind to human recombinant PDE3B protein(the main subtype of PDE in adipocytes),and its kinetic parameter Ka and Kd values are 8 M-1s-1 and 0.69 s-1,respectively.Similarly,in enzyme activity assay,salicylate sodium also directly inhibited the activity of PDE3B(IC50=13.81 mM).In the PDE3B-deficient adipocytes,the inhibitory effect of salicylate sodium on MCP-1 disappeared.And two other inhibitors of PDE3,milrinone and cilostamide,have similar effects to salicylate sodium.Moreover,salicylate sodium also directly inhibited the activity of PDE3A(IC50=6.0 mM)and PDE4B(IC50=0.81 mM),indicating that salicylate sodium is a non-selective PDE inhibitor.In conclusion,this study identifies a novel target of salicylate sodium in the treatment of metabolic inflammation.PDE3B.In TNF-α-stimulated adipocytes,it can directly inactivate PDE3B to elevate the intracellular cAMP level,which leads to the activation of PKA and subsequently increases the MKP-1 expression.The latter effect can dephosphorylate the activated ERK and p3 8,thus resulting in the decrease of MCP-1 production.These effects of salicylate sodium are independent of its previously identified targets,IKKβ and AMPK.This study provides the evidence for a hitherto unknown anti-inflammatory pathway of salicylate sodium in obesity-related inflammation and proposes a rationale for the application of its prodrugs in metabolic diseases.