The Effect Of SGLT-2 Inhibitor Empagliflozin On Ischemic Brain And Related Mechanism Study In Mice

Objective: Stroke is a serious cerebrovascular disease which can be devided as hemorrhagic and ischemic stroke.Ischemic stroke account for approximately 65%-80%of all stroke cases.Stroke has become one of the main causes of death and disability worldwide.However,due to the complex pathogenesis of ischemic stroke,there are very limited therapeutic drugs available,and these drugs are compromised by narrow therapeutic time-window and many adverse effects.Despite the facts that some drugs ameliorate neurological dysfunction,few of the drugs can significantly improve neurological functions and the quality of life of patients after stroke.Accordingly,it is urgent to find more effective treatment drugs for stroke.Hyperglycemia is a common complication of ischemic stroke,and the extension of blood glucose increase after stroke is closely associated with the poor prognosis and mortality in stroke patients.Previous studies have shown that controlling stroke blood glucose can improve the prognosis of stroke.Insulin injection remains the most frequent prescription to control hyperglycemia after ischemic stroke.Nevertheless,insulin may induce hypoglycemia which may further accelerate ischemic brain injury.Therefore,it is essential to seek out a rapid hypoglycemic drug that can replace insulin.Sodium-dependent glucose transports 2(SGLT-2)is a glucose transporter located in renal tubular epithelial cells and is the main protein involved in renal glucose uptake.SGLT-2 inhibitors are a new class of anti-diabetic drugs by inhibiting the reabsorption of glucose by the kidneys and promoting the elimination of glucose from the urine.Empagliflozin is an SGLT-2 inhibitor,in addition to lowering blood glucose,it can also reduce blood pressure by reducing arteriosclerosis,and improve heart and kidney functions by changing energy supply.The above-mentioned pharmacological effects imply that empagliflozin could be a potential drug for stroke therapy.The present research aims to clarify the potential protective effect of empagliflozin on ischemic brain injury and neurological dysfunction.We further explored the its mechanism underlying neuroprotection.Methods: C57BL/6 mice were subjected to photo-thrombosis model,the mice were treated with empagliflozin 3,10,30 mg/kg by gavage everyday,and the mouse blood glucose was recorded every 3 hours after the next day(1,3,5,and 7 days).Indicated dosage of empagliflozin was administrated for either 7 or 14 consecutive days,and the grid-walking task and the cylinder task were performed daily to detect the motor function of the mice.Alternatively,the mice were treated with empagliflozin simultaneously with 10% glucose(i.p.)for seven consecutive days after the photo-thrombosis model to test whether glucose can reverse the effect of empagliflozin.After the permanent middle cerebral artery occlusion(pMCAO)or tMCAO(transientMCAO)surgery to establish an animal model of acute cerebral ischemia.Empagliflozin(10 and 30 mg/kg)was administered to mice one hour after the onset of occulusion.Braininfarct volume and neurological defect score were assayed 24 hours after surgery.Toluidine blue staining was used to detect the infarct volume of the mouse 7days after the photo-thrombosis model was established,and the immunofluorescence experiment was used to detect the number of neurons around the infarct area;the microglia of the infarct area were detected 7 days after the photo-thrombosis.The number and microglial protrusions are used to detect the activation of microglia.Result: The blood glucose elevation after photo-thrombosis significantly decreased with empagliflozin administration and can be maintained for 18-21 hours.after the photo-thrombosis surgery,the will be given by gavage for 14 consecutive days and 7 days.Treatment of empagliflozin(10 mg/kg)for consecutive 7 or 14 days significantly decreased the rate of false foot in grid-walking task and the assymetric index in cylinder task.At the dose of 30mg/kg,however,empagliflozin even aggravated photo-thrombosis-induced neurological dysfunction,whilet the dose of3mg/kg showed no effect.Unexpectedly,the protective effect of empagliflozin(10mg/kg)could not be reversed by glucose treatment,suggesting that the empagliflozin-conferred neuroprection may not related with its effect on controlling blood glucose.Gavage of empagliflozin one hour after tMCAO surgery could not significantly reduce the brain infarct volume and neurological dysfunction.Moreover,empagliflozin even significantly increase brain infarction and neurological defect scores after pMCAO surgery.The results of immunofluorescence showed that empagliflozin(10 mg/kg)showed no significant benefits on the infarct volume and neuronal loss in the ischemic area after the photo-thrombosis operation.Empagliflozin(10 mg/kg)significantly alleviated the microglia activation in the ischemic area.Conclusions: Empagliflozin alleviated ischemia-induced neurological dysfunction with consecutive administration,and its pharmacodynamics is dose-dependent.Empagliflozin-conferred neuroprotection may not relate with its effects on lowing blood glucose,alternatively,empagliflozin may play a neuroprotective effect by inhibiting the excessive activation of microglia in ischemic brains.Empagliflozin cannot protect against acute ischemia-induced brain injury in mice.