| BackgroundDiabetic peripheral neuropathy(DPN)is a group of heterogeneous diseases caused by long-term hyperglycemia and damage innervous system in diabetic patients lead to neuronal dysfunction,and often presenting as distal symmetric multiple diabetic neuropathy with pain,feelingnumbness,ant mobility,burning,which can make the patient live in pain for a long time,affect the quality of life.Because its pathogenesis has not been fully elucidated,effective treatment methods are often lacking.Dapagliflozin,a new type of hypoglycemic drug,is concerned because it is not dependent on the hypoglycemic mechanism of insulin secretion.Dapgliflozin reduces the renal glucose threshold by inhibiting the SGLT-2 protein reabsorption of urine glucose in the proximal renal tubules,thereby expelling excess glucose to achieve the goal of reducing glucose levels in blood circulation.In addition to the hypoglycemic effect,dapagliflozinalso influence blood glucose,blood lipid,body weight and other factors,which have the effects such as lowering blood pressure,reducing body weight.The effect of reducing body weight and improving blood lipid metabolism is mainly related to the loss of energy caused by reducing the reabsorption of urine glucose,which is equivalent to a certain degree of energy limitation.Studies have shown that energy limitations can protect neuron and be used to treat nerve-damaging diseases such as epilepsy,Alzheimer's disease,spinal cord trauma,etc.In addition to reducing blood glucose,whether there is neuroprotective effect on the nervous system and its molecular mechanism is the scientific problem in this experiment.PurposeThis experiment used the PDN rat model,and PDN rats were treated with dapagliflozin to observe its behavior changes,and to detect the analgesic effect of dapagliflozinon PDN rats.Detect the protein expression changes of histone deacetylase HDAC2 and the potassium channel Kv1.2 in rat dorsal root ganglion after dapagliflozin treatment and explore the molecular mechanism of analgesic effect of dapagliflozin.Methods1.The diabetic model rats was established used STZ.Blood glucose was measured on the D3,D7,D14 after operation.Detected the mechanical pain,cold pain,heat pain threshold and screening out PDN rats.2.When the mechanical pain threshold decreased by 50% on the D14 after operation,the rats were divided into five groups:naive group,STZ+vehicle group,STZ+1mg/kg dapagliflozin group,STZ+5mg/kg dapagliflozin group,STZ+10mg/kg dapagliflozin group,and measured the changes of mechanical paw withdrawal threshold,thermal paw withdrawal latency and cold paw withdrawal latency at D3,D7 and D14 after treatment with dapagliflozin.3.?-hb levels in serum were assessed using a ?-hb ELISA kit at the 14 days afterusing dapaglliflozin.4.On the D14 after treatmented with dapagliflozin,usewestern-blot method to compare the protein expression levels of Kv1.2 and HDAC2 in rats' DRG tissues.5.Use immunofluorescence tecniques to detect the expression changes of HDAC2 and Kv1.2 proteins in three groups of rats in DRG tissue.6.PDN rats were divided into three groups at D14 of STZ injection: naive,PDN+vehicle,PDN+romidepsin,injected the rats withromidepsin,a specific HDAC2 inhibitorfor 2 weeks,anddetect their behavioral changes at D3,D7 and D14 after injected romidepsin.7.Expression of Kv1.2 protein in DRG wasdetected after injection of specific inhibitors romidepsin in rats for 14 days.8.Use 1mg/kg,5mg/kg,and 10mg/kg dapagliflozin in CCI rats,the effect of dapagliflozin therapy on pain threshold was observed at D3,D7,D14 after use dapagliflozin.9.The effect on HDAC2 protein expression in DRG was detected by Western blot after dapagliflozin treatment in CCI neuropathic pain rats.10.Use PC12 cells for cell culture,the cells were divided into two groups,one group was cultured in normal DMEM medium,the other group was cultured with DMEM medium added ?-hb.Collecte the cells 24 hours later for western-blot to detect the expression of HDAC2 and Kv1.2 protein;Assess the regulatory effect of ?-hb on HDAC2 at the cell level.Results1.Compared with the naive group,the blood glucose of diabetic rats increased significantly on the D3 after operation(***p<0.001),and remained high level every week after operation which noted that diabetic rats model were successfully established.The mechanical pain threshold of 70% diabetic rats decreased significantly after 14 days(***p<0.001).The pain threshold of rats in naive group was stable at the normal level,which proved that the diabetic neuropathic pain model was successfully established.2.The changes of blood glucose and mechanicalpaw withdralwal threshold,thermalpaw withdralwal latency,cold paw withdralwal latency were observed at D3,D7,D14,after medication.We found that the blood glucose decreased after dapagliflozin was used in PDN rats,and compared with PDN+vehicle group rats themechanicalhyperalgesia(#p < 0.05)?cold hyperalgesia(##p<0.01)?heat hyperalgesia(#p < 0.05)of PDN+5mg/kgdapagliflozin and PDN+10mg/kg group rats relieved at D7 after medication,and the pain state of vehicle group of rats did not relieve.3.The ?-hb levels inserum of three groups of rats were detected using a ELISA kit.The results showed that the ?-hb levels of rats in DPN+dapagliflozin group was increased obviously compared with PDN+vehicle grouprats(###p<0.001).4.The protein expression of HDAC2 and Kv1.2 was detected D14 after dapagliflozin administration(i.e D28 after STZ injection).The results showed that the Kv1.2 protein level in the DRG tissues of PDN rats was significantly decreased(**p< 0.01)and the HDAC2 protein level was significantly increased(***p<0.001)compared with that naive group.The Kv1.2 level of rats in PDN+10mg/kg dapagliflozin group was recovered(#p< 0.05)and HDAC2 in PDN+10mg/kg dapagliflozin group decreased(##p<0.01)compared to PDN+vehicle group rats.5.The protein expression level of Kv1.2 and HDAC2 in DRG neurons in three groups of rats was detected by immunofluorescence tecniques.The results showed that compared with naive group,theaverage flourscence indensity of Kv1.2 positive cells decreased(***p< 0.001)and the average flourscence indensity of HDAC2 positive cells increased(***p<0.001)in the DPN+vehicle group.After using dapagliflozin for 14 days,theaverage flourscence indensityof Kv1.2 positive cells increased(###p < 0.001)and the average flourscence indensity of HDAC2 positive cells decreased(###p<0.001)compared with the DPN+vehicle group.6.PDN rats injected with HDAC2 specific inhibitors romidepsin and detected behavioral changes,the results showed that mechanical(###p<0.001),heat(#p<0.05)and cold(###p<0.001)pain sensitivity were alleviated.7.Use western blot to test the expression of protein,the results showed that kv1.2 protein expression recovered(##p<0.01)after using inhibitors compared with PDN rats injecting saline.8.Use CCI neuropathic pain rats model.Treat the rats with different doses of dapagliflozin for 14 days,detect the pain threshold at D3,D7 and D14,the results showed that the mechanical paw withdralwal threshold(#p< 0.05),thermal(#p<0.05)paw withdralwal latency were recovered to a certain degree.9.The expression of HDAC2 proteindecreased(#p<0.05)in L4-L6 DRG tissues of rats in CCI+dapagliflozin group compared with CCI+vehicle group.10.Collected the cells for western blot,the results showed that the expression level of Kv1.2 protein increased(*p<0.05)and the expression level of HDAC2 protein decreased(*p<0.05)in the DMEM+?-HB media group compared with the DMEM medium group.ConclusionIt can concludethat dapagliflozin can relieve neuropathic pain of DPN and CCI model rats.The mechanism may be that dapagliflozin increased the content of ketone body ?-hb in vivo and inhibited the expression of HDAC2,thus flipped the low expression of Kv1.2,reduced the excitability of neurons and relieved pain. |
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