Mechanistic Studies On Salsalate Effect On Metabolic Disorders In A Mouse Model Of DIO

Salsalate is an ester formed by two salicylic acid molecules,which has anti-inflammatory properties.It has beneficial effect against metabolic disorders in clinical trials.But the mechanism of regulating and improving the body’s metabolic process by salsalate is unclear.AMPK is the guardian of metabolism and mitochondrial homeostasis and monitors energy balance at the cellular levels.Energy balance plays a central role in human diseases such as type 2 diabetes,cancer and inflammatory disorders.This study aims to determine the relationship between salsalate and AMPK pathway,and to explore its mechanism in the treatment of metabolic diseases.We first used C57BL/6J mice to construct a mouse model of DIO,and then treated with 300 mg/kg salsalate by intraperitoneal injection or with a vehicle for 7 consecutive days to test the therapeutic effect of salsalate.We found that in DIO mice,salsalate treatment can significantly improve metabolic indicators,including decreased body weight gains,reduced white adipose tissue mass,improved glycemic control,and lowered levels of hepatic triglycerides.Histology analysis revealed that salsalate treatment reduced the size of fat cells and the infiltration of macrophages in obese adipose tissue,and decreased hepatosteatosis,including both macrovesicular and microvesicular steatoses.We analyzed adipose tissue macrophages infiltration by FACS studies to determine whether salsalate treatment had an effect on macrophages infiltration.It was found that salsalate treatment profoundly decreased F4/80~+CD11b~+macrophage infiltration,which was reduced by 43%compared with the untreated group(P<0.01).FACS studies were carried out to quantitatively investigate salsalate effect on macrophage infiltration to the livers by staining for F4/80~+CD11b~+positive cells.Salsalate treatment significantly decreased the number of F4/80~+CD11b~+cells,which was reduced by 57%compared with the HFD group(P<0.01).It shows that salsalate reduced the macrophage infiltration of obese adipose tissue and liver.Salsalate on AMPK activation was studied by immunoblotting for AMPK and ACC phosphorylation in animals.AMPK activation was profoundly repressed in HFD fed mice.Salsalate treatment partially restored AMPK phosphorylation,accompanied by reduced caspase-6 activity and cleavage.Enzymatic assay and cell culture studies showed that salsalate promoted AMPK activation by directly activating AMPK.Then we tested the stability of the prepared salsalate.The HPLC analysis results showed that the salsalate is relatively stable in aqueous solutions with neutral p H,which excluded the possibility that the salsalate effect on the experiment was associated with salicylic acid production from salsalate hydrolysis.The concentration of salsalate to promote significant phosphorylation of AMPK,as well as ACC protein,was detected at approximately 0.25-0.5 m M in cultured cells.We found that salsalate at 5 m M promoted significant phosphorylation of AMPK and ACC.Salsalate promoted AMPK activation was detected through the course of the treatment from 2 to 24 hours.The effect of salsalate on AMPK phosphorylation was not restricted to HEK293T cells since the effect was also detected using hepatocellular carcinoma Hep G2 cells.Gene expression was detected by real time PCR.Next,we detected the gene expression of proinflammatory response and adipogenesis in WAT by q PCR.Compared with the HFD group,the levels of IL-1β,IL-6,and TNFαm RNA in the adipose tissue from the salsalate-treated group were significantly decreased by approximately 65.1%,88.2%,and 69.1%respectively(P<0.01).Salsalate also obviously inhibited the gene expression of adipogenesis such as PPARγ,C/EBPa,SREBP-1c and a P2,and m RNA levels were reduced by 45.8%,59.1%,56.3%,and 42.9%respectively(P<0.05).In summary,salsalate can significantly improved the metabolic disorders in obese mice.Its regulatory effect is related to the phosphorylation of AMPK and the inhibition of caspase-6,thereby reducing the infiltration of macrophages in obese adipose tissue and liver,decreasing body weight gains and improving glycemic control in DIO mice.This study firstly uncovers the salsalate can directly activate AMPK enzymatically,and can improve metabolic disorders through the AMPK-caspase-6 axis.This study therefore clarifies the improvement effect of salsalate on metabolic disorders and its potential mechanism,providing a theoretical basis for the development of treatment methods for metabolic diseases.