Effects And Mechanisms Of Soluble Epoxide Hydrolase Inhibitor TPPU On Improving Diabetes And Hypertension

Objective:Diabetes and hypertension are major health problems worldwide,and when they coexist,they will significantly increase the incidence and mortality of patients with cardiovascular events.Previous studies have shown that Epoxyeicosatrienoic acids（EET）/Soluble epoxide hydrolase（sEH）system exerts protective effects on diabetes or hypertension,but the effects and mechanisms on these comorbidities of the two diseases remain unknown.Therefore,this study aims to investigate whether the sEH inhibitor TPPU（（Trifluoromethoxyphenyl-3-（1-propionylpiperidin-4-yl）urea））can reduce blood pressure and blood glucose,and improve insulin resistance in mice with diabetes and hypertension.If TPPU can play a protective role in lowering blood pressure and lowering blood sugar,combined with previous studies,it has been found that TPPU can reduce blood glucose by inhibiting the expression of sodium-glucose cotransporter 2（SGLT2）in the kidney in db/db mice.This study will further explore whether TPPU can reduce blood pressure and blood glucose in diabetic and hypertensive mice by inhibiting the expression of SGLT2 in the kidney and its specific mechanism.Methods:Animal experiment:（1）6-week-old C57BL/6 mice were fed with either a standard chow/normal salt（SC-NS,n=24）diet or a high fat and high salt（HF-HS）diet,the high-salt and high-fat diet was used to construct a model of diabetes and hypertension.After 4 weeks of diet intervention,the sEH inhibitor TPPU or sodium-glucose cotransporter 2（SGLT2）inhibitor dapagliflozin was infused into the mice by drinking water.The mice were divided into the following five subgroups:（1）SC-NS group（2）SC-NS+TPPU group（3）HF-HS group（4）HF-HS+TPPU group（5）HF-HS+Da group.（2）The body weight,blood glucose and blood pressure were detected monthly,the GTT and ITT tests were used to evaluate glucose tolerance and insulin resistance,the metabolic cages were used to detect metabolic activities and collect urine for 24 hours before the end of the experiment.（3）At the end of the experiment,the mice were sacrificed,and blood and urine samples were collected,and tissue samples such as kidney,fat,aorta,liver,pancreas,and skeletal muscle were also collected.（4）The morphology of adipose and kidney were evaluateby HE staining.（5）The expression of sEH and SGLT2,and the expression of IKKα/β/NF-κB pathway related molecules in kidney were detected by Western Blot and immunohistochemistry.（6）The enzyme-linked immunosorbent assay（Elisa）was used to detect the activity of sEH and the concentration of 14,15-EET in kidney.（7）Spectrophotometry was used to detect the concentration of glucose and sodium ion in urine.Cell experiment:Palmitic acid and sodium chloride were used to interfere renal tubular epithelial cells（HK-2）to simulate the internal environment of diabetic and hypertensive animals.sEH inhibitors TPPU,EET,and EPHX₂siRNA were used to treat HK-2 cells,and Western Blot was used to detect SGLT2 and pathway IKKα/β/NF-κB pathway related molecules expression.Immunofluorescence was used to detect the nuclear localization of NF-κB.Results:1.The initial body weight of each group were equivalent.After 12 weeks of diet intervention,the weight,epididymal fat mass,random and fasting blood glucose,systolic blood pressure,diastolic blood pressure,mean arterial pressure of HF-HS group mice was significantly higher than SC-NS group.IGTT test showed that HF-HS group had obvious impaired glucose tolerance compared with SC-NS group.ITT test showed that HF-HS group had obvious insulin resistance compared with SC-NS group.These results show that the intervention of high-salt and high-fat diet induces hypertension and insulin resistance in mice.2.The results of Western Blot and immunohistochemistry showed that compared with the SC-NS group,the expression of sEH and SGLT2 in the kidney of the HF-HS group was significantly up-regulated.3.In order to further explore the role of sEH and SGLT2 in hypertension and diabetes,the metabolic indicators of each group of mice were tested.The results showed that:TPPU and dapagliflozin improved the fasting blood glucose and decreased systolic blood pressure in HF-HS group,TPPU also decreased the diastolic blood pressure and mean arterial pressure of mice in HF-HS group.TPPU and dapagliflozin improved the abnormal glucose tolerance and insulin resistance in mice caused by a high-salt and high-fat diet.The results of the metabolic cage showed that the high-salt and high-fat diet significantly reduced the respiratory exchange rate（RER）of mice,suggesting that the high-salt and high-fat diet promoted the shift of substrate selection from glucose to lipid.The intervention of TPPU and dapagliflozin can reverse the changes in substrate selection caused by a high-salt and high-fat diet.These results indicate that both the sEH inhibitor TPPU and the SGLT2 inhibitor dapagliflozin can effectively improve hypertension,insulin resistance,and abnormal glucose tolerance caused by a high-salt and high-fat diet,and can reverse the substrate selection caused by a high-salt and high-fat diet.4.The high-salt and high-fat diet increased the urine volume,urine glucose and urine sodium excretion,and the intervention of TPPU and dapagliflozin further increased the urine volume,urine glucose and urine sodium excretion.Western Blot and immunohistochemical results showed that TPPU intervention inhibited the up-regulation of SGLT2 in mouse kidneys caused by an HF-HS diet.Elisa showed that a high-salt and high-fat diet increased renal sEH activity and reduced 14,15-EET level.TPPU intervention can significantly inhibit renal sEH activity,thereby increasing the kidney 14,15-EET level in mice.These results suggest that TPPU inhibits the reabsorption of sodium and glucose by increasing the level of kidney 14,15-EET and then inhibiting the expression of SGLT2,thereby reducing blood pressure and blood glucose,and improving insulin resistance.5.Western Blot and immunohistochemical showed that an HF-HS diet increased infiltration of macrophages in mice kidney,and increased expression and activation of of IL-1β,MCP-1,TNF-αand NF-κB,as well as activated IKKα/β/NF-κB pathway.TPPU intervention can reduce renal macrophage infiltration,inhibit the expression of inflammation-related molecules and inhibit IKKα/β/NF-κB pathway activation.6.Cell experiment show that TPPU and 14,15-EET can significantly inhibit palmitic acid and sodium chloride induced upregulation of SGLT2 in HK-2 cells,and 14,15-EET inhibits the activation of IKKα/β/NF-κB pathway in a dose-dependent manner.The up-regulation of SGLT2 in renal tubular epithelial cells induced by palmitic acid and sodium chloride,an inhibitor of NF-κB,BAY 11-7082 inhibited the upregulation of SGLT2 in HK-2 cells induced by palmitic acid and sodium chloride,confirmed the regulatory effect of NF-κB on SGLT2.Conclusion:sEH inhibition alleviated insulin resistance and hypertension induced by a high-fat and high-salt diet in mice via increased urine glucose and sodium excretion mediated by decreased renal SGLT2 expression,which was due to inactivation of IKKα/β/NF-κB induced inflammatory response.