Accumulation Of Salicylic Acid Based Mechanism And Tetramethylpyrazine Intervention Study Of Aspirin Resistance In Diabetic Mice

BackgroundAspirin is the cornerstone of anti-platelet therapy in the secondary prevention for stroke,myocardial infarction and other cardiovascular events.However,patients continue to experience atherothromboembolic events despite aspirin therapy,which is known as "aspirin resistance(AR)".Diabetes is an established risk factor of AR which presents a challenge to clinical anti-coagulation therapy,but the key mechanisms remain elusive.The invigorating blood circulation and eliminating stasis in traditional Chinese medicine has been widely applied to clinical antiplatelet and anticoagulanttherapy,Chinese medicine active ingredient has become a source of therapeutic drug candidate against AR.Objective1.To assess antiplatelet effect of aspirin on gender differences in diabetic state.2.To uncover the potential link and possible mechanism between pathogenic accumulation of SA,the major metabolite of aspirin,and AR in diabetic state.3.To discuss metabolic regulation associated with AR in T2DM and the intervention by TMP from the perspective of endogenous metabolite changes.MethodsHigh-fat diet and streptozotocin(HFD/STZ)induced Type 2 diabetes mellitus(T2DM)mice model.Normal mice(male,n=14;female,n=14)were randomly divided into two groups and treated with 0.5%CMC-Na or aspirin dissolved in 0.5%CMC-Na for 10 days,respectively;T2DM mice were divided into five groups and treated with vehicle(0.5%CMC-Na)for 10 days(male,n=7;female,n=7),aspirin(13 mg/kg,i.g)(dissolved in 0.5%CMC-Na)for 10 days(male,n=7;female,n=7),aspirin for 1 day followed by 9 days of SA(10 mg/kg,i.g,equal molar concentration of aspirin)(female,n=7),NaHC03(600 mg/kg,i.g)plus aspirin for 10 days(female,n=7),or tetramethylpyrazine(TMP)(100 mg/kg,i.p)plus aspirin for 10 days(female,n=7),respectively.The levels of blood glucose(BG)?Insulin(INS).cholesterol(TC).triglycerides(TG)Were measured by enzymatic method and the pathological changes in the renal tissues were examined by optical microscopy.IL-1? and IL-6 in mcie serum were detected by ELISA.Platelet function was analyzed using Flow cytometry analysis of platelet P-selectin(CD62P).TXB2 and 6-keto-PGF1? contents were determined by radioimmunoassay and the concentration of SA in plasma was measured with a high-performance liquid chromatography(HPLC).QRT-PCR analysis was performed to detect the renal organic anion transporters(OATs),sodium-coupled monocarboxylate transporters(SMCTs)anduric acid transporter 1(URAT1).The expression of cycloxygenase-2(COX-2)was detected by immunofluorescence staining and qRT-PCR.Gas chromatography/mass spectrometer(GC/MS)coupled with multivariate statistical analysis was used to identify the alteration of global serum metabolites associated with T2DM mice as compared to healthy controls and responses to aspirin or TMP plus aspirin.Results1.Impaired aspirin responsiveness in T2DM female miceHFD/STZ induced T2DM mice were characterized by increased body weight,hyperglycemia and hyperlipidemia,although plasma insulin levels were mildly elevated,suggesting the development of insulin resistance.The expression of platelet CD62P,a well-established marker for platelet activation was significantly increased in T2DM females.Aspirin treatment significantly decreased the expression of platelet CD62P in control females but was ineffective in T2DM counterparts,suggesting the emergence of High on-aspirin platelet reactivity(HAPR).As expected,aspirin decreased the ratio of TXB2/6-keto-PGF1? in control but not in T2DM females.Of note,no impairment of aspirin responsiveness was observed in the T2DM male mice.2.Salicylic acid renders aspirin resistance via abrogating the compensatory upregulation of endothelial COX-2 in diabete and downregulation of various SA efflux transporters explains SA accumulationIt was of interest to note that SA was only accumulated in female T2DM mice after continuous dosing of aspirin,as evidenced from either trough plasma concentration determined on day 1,5,and 10 or plasma pharmacokinetic profile determined on the last day.Pre-administration with SA increased both platelet CD62P expression and TXB2/6-keto-PGF1? ratio in female T2DM mice,while pretreatment with NaHCO3 caused the opposite effect.Correlation analysis showed that SA concentration was positively correlated with TXB2/6-keto-PGF1?in T2DM female mice.High-level SA in the circulation abrogates the compensatory upregulation of endothelial COX-2 in diabetic mice,decreases the production of the protective PGI2,thereby precipitating the platelet activation toward aggregation.The retention of SA partially due to impaired renal excretion by organic anion transporterl and 3(OAT1,OAT3),odium-coupled monocarboxylate transporter 1(SMCT1)uric acid transporter 1(URAT1)and acidotic condition.3.TMP-Treated AR in diabetic state with the related regulation of metabolic pathwaysWe were able to identify 79 of the 120 metabolites inthe mice serum using our optimized GC/MS analysis protocol.From the PLS-DA scores plot,the control group was able to separate from aspirin trementment group Significantly.However,the T2DM group was not able to separate from aspirin treatment group,indicating that the failure of the aspirin on diabetes treatment.Altered serum metabolites in T2DM female mice include increased Pyruvic Acid?3-Hydroxybutyric Acid?4-Hydroxybutyric Acid?Arachidonic Acid?Glucose?D-Turanose?Cholesteroland decreased D-Lactic Acid?Isoleucine?L-Serine?L-Threonine?Tryptophan.Aspirin can not only reverse the changed endogenous metabolitesbut broaden the metabolic difference.However,TMP treatment was able to reverse more abnormal levels of metabolites in T2DM female mice after aspirin,such as Arachidonic Acid?Glucose?Pyruvic Acid?Cholesterol?Uric acid and Urea?suggesting that it is more efficient to alter the Arachidonic acid metabolism?Pyruvate metabolism?Glycolysis or Gluconeogensis?TCA cycle?Tryptophan metabolism of T2DM-associated AR.ConclusionsIn summary,our study suggests that the retention of SA upon long term treatment of aspirin may represent an important determinant of AR in the female diabetic mice.Mechanistically,SA suppresses the inflammation triggered adaptive upregulation of endothelial COX-2,break the functional balance of COX-1/COX-2,thereby decreasing the production of the protective PGI2.Through the analysis of the metabonomics,TMP is more efficient to alter the metabolism of T2DM-associated AR.Findings from our study suggest that targeting impaired SA excretion and combined therapy with TMP may serve a novel intervention strategy on T2DM-associated AR.