The Study Of Saxagliptin On Diabetes With NAFLD Model Via CaMKK?/AMPK Pathway

Objective:Non-alcoholic fatty liver disease(NAFLD)is a major hepatic manifestation of metabolic disease and an important complication of diabetes mellitus(DM).Inflammation,immunity,insulin resistance(IR),oxidative stress,as well as endoplasmic reticulum stress(ERS)and apoptosis are involved in the progression of NAFLD.Notably,inflammation plays a crucial role in the progression of NAFLD.Moreover,the pathological manifestation of NAFLD is closely related to accumulation and polarization of hepatic macrophages.DPP-4 inhibition broadly applied in clinical as an antidiabetic agent,leading to glucose-dependent hypoglycemic effect due to increased incretin peptides and subsequent incretin secretion.Besides,Dipeptidyl peptidase-4(DPP-4)is considered as a key factor in immunity and inflammation,thus DPP-4 inhibition played extra-pancreatic effects via other certain mechanisms.Presently,the effect of DPP-4 inhibition on NAFLD has been poorly reported and underlying mechanisms remain uncertain.Meanwhile,saxagliptin,a newly DPP-4 inhibition,has not been fully studied yet especially on NAFLD.Based on inflammatory effect of DPP-4,we establish diabetic rats model and cell culture to study whether saxagliptin can suppress hepatic inflammation of NAFLD and if it is mediated by macrophage accumulation and polarization.Moreover,we prefer to illustrate the pathway involved in in vivo and in vitro.Methods: 1.In vivo: 45 clean Sprague�Dawley(SD)rats were purchased for building diabetic model and fed standard chow for one week,Firstly,we chosen 15 rats at random still fed standard rodent diet as normal control group,while the rest were fed high-fat diet.After a period of 10 weeks feeding,we measured body weight,blood glucose and lipid and performed IPGTT,while 30 high-fat diet rats were then injected with a single low dosage of STZ for establishing diabetes model.Randomly,15 well-established diabetic rats were treated with saxagliptin(10mg/(kg�d))via gastric gavage for 12 weeks while control groups were treated with equal normal saline for 12 weeks.We finally subdivided the above rats into three groups: normal control group(NC groups),model group(DM groups),drug treatment group(DM+SAX groups).After measuring body weight,blood glucose and collecting blood sample at the end of 12 th week,we sacrificed all rats and harvested the livers.Then,we use Haematoxylin and eosin(H&E)staining for histopathological analysis of livers.We apply Triglyceride and Cholesterol assay Kits for liver lipid content and DPP-4 assay kit for DPP-4 activity measurement.Further,we examined expression of NF-?B,TNF-?,IL-1? by immunohistochemistry technique and expression of DPP-4,CD68,i NOS and CD206 by immunofluorescence microscopy.We determined m RNA levels of NF-?B,TNF-?,IL-1? by q RT-PCR and protein levels of DPP-4,NF-?B,TNF-?,IL-1?,IL-6,CD68,i NOS,CD206,Ca MKK?,p-AMPK by Western blotting.2.In vitro: Human THP-1 monocyte line was one of the most commonly models for studying macrophage.Firstly,THP-1 monocytes were differentiated into M0 macrophages using 50ng/ml phorbol 12-myristate 13-acetate(PMA).Then M0 macrophages were stimulated into M1 macrophages with LPS and M2 macrophages with IL-4.We detected M1 marker(i NOS)and M2 marker(CD206)by immunofluorescence microscopy to ensure successful polarizaiton of M1/M2 macrophages.We added saxagliptin(5?mol/L)and Ca MKK? inhibitor STO-609(10?M)and grouped for intervention.Then we detected protein levels of p-AMPK,NF-?B,TNF-?,CD206 by Western blotting to explore the effect of saxagliptin on inflammation and Ca MKK?/AMPK pathway in vitro.Results: 1.After 10-week diet,in high-fat diet group,weight and lipid level were obviously increased(P<0.05).Blood glucose was higher but no significant(P>0.05).IPGTT showed higher blood glucose after glucose load in high fat diet,and the calculated AUC was also significantly increased(P<0.05).2.After 12-week treatment by gavage,DM group and DM+SAX group displayed significant low-body weight versus NC group(P<0.05),while no difference significantly occurred between DM and DM+SAX groups(P>0.05).Significantly,DM group showed higher levels of blood glucose,lipid than NC group(P<0.05),while no markedly declination were found in DM+SAX group versus DM group(P>0.05).The liver function didn't alter among the three groups(P>0.05).but BUN in DM group was higher than NC group,and significantly decreased by saxagliptin versus DM group(P<0.05).3.Haematoxylin and eosin(H&E)staining for histopathological analysis of livers: In NC group,regular structure of hepatic lobules and uniform morphology of hepatocytes arranging in cords were observed.DM group was characterized by disordered hepatic lobules and enlarged hepatocytes which were accumulated with many vacuolar-like lipid droplets.While in DM+SAX group,hepatocytes with obvious decreased lipid were arranged in regular cords.4.The lipid content in liver of each group: DM group showed significantly higher TG and TC levels than NC group(P<0.05).When treated saxagliptin,the level of them significantly decreased with statistical difference(P<0.05).5.DPP-4 activity and expression levels of livers: DPP-4 activity of DM group was significantly enhanced versus NC group(P<0.05).DM+SAX group showed significant inhibition of DPP-4 activity in contrast to enhanced DPP-4 activity in DM group(P<0.05).DPP-4 protein expressions showed the same trend detected by western blotting and immunofluorescence analysis(P<0.05).6.Expression levels of hepatic inflammatory factors: Compared with NC group,marked increase of inflammation-related factors NF-?B,TNF-? and IL-1? were observed in DM group while obvious reduction was shown in DM+SAX group by Immunohistochemistry technique.Consistently,the relative m RNA and protein levels of all above were quantified to have significant suppression in DM+SAX group using q RT-PCR and western blotting(P<0.05).7.Macrophage infiltration and macrophage phenotypes in each group: Compared with NC group,DM group increased the infiltration of total-macrophages(CD68),especially accumulation of pro-inflammatory M1 phenotype,with the decreasing immunoreactivity of M2 phenotype by Immunofluorescence Microscopy.When treated with saxagliptin,inverse images were observed in liver sections.Similarly,the relative protein levels of above markers exhibited the same trend significantly by western blotting(P<0.05).8.Expression levels of Ca MKK?/AMPK of livers: Using Western blotting,protein level of Ca MKK? and p-AMPK was dramatically decreased in DM group compared with NC group(P<0.05),while Ca MKK? and p-AMPK showed higher level in DM+SAX group versus DM group(P<0.05).9.Polarization of M1/M2 macrophages in vitro: THP-1 monocytes were differentiated into resting macrophage(M0)with PMA.95% monocytes were adhered and in an irregular cellular pseudopod extend at the concentration of 50ng/ml and 100ng/ml PMA for 48 h.Avoiding side effect of PMA,we finally choose 50ng/ml PMA for differentiation.Then,we stimulated M0 macrophage into M1 and M2 macrophages with 1mg/ml LPS or 40ng/ml IL-4 respectively.The immunoreactivity of i NOS was significantly increased in PMA+LPS group.While CD206 showed obvious immunoreactivity in PMA+IL-4 group.Consequently,M1/M2 macrophages were polarized successfully.10.The effect of saxagliptin on Ca MKK?/AMPK mediated M1/M2 macrophage-regulated inflammation: Using western blotting,we determined protein levels of p-AMPK,NF-?B and TNF-? in M1 macrophage and p-AMPK,CD206 in M2 macrophage treated with saxagliptin and STO-609 or not.Saxagliptin significantly increased protein level of p-AMPK both in M1 and M2 macrophage,while was significantly suppressed by STO-609(P<0.05).Moreover,protein level of NF-?B and TNF-? were reduced by saxagliptin in M1 macrophage,while were markedly elevated by STO-609(P<0.05).Then we found CD206 expression was significantly promoted by saxagliptin in M2 macrophage and was suppressed by STO-609 with decreasing trend.(P>0.05).Conclusion: 1.Saxagliptin can attenuate liver steatosis in STZ-induced diabetic model.2.Saxagliptin can suppress hepatic inflammation beyond hypoglycemic effect.3.Saxagliptin can regulate macrophage polarization by inhibition of pro-inflammatory M1 phenotype and enhancement of anti-inflammatory M2 phenotype.4.Saxagliptin maybe via Ca MKK?/AMPK in the regulation of M1/M2 macrophage,mainly in the inhibition of M1 macrophage.