Purinergic Receptor Mediated Diabetic Peripheral Neuropathy And Its Possible Mechanism

Background: Diabetic neuropathy is the most common and difficult complication of diabetes.It is the highest incidence rate and mortality rate of diabetes,which brings enormous economic burden to treatment of diabetic patients.Neuropathy occurs in 50% of adult diabetic patients.Diabetic hyperglycemia can cause peripheral nerve damage by inducing ischemia,oxidative stress and inflammation.Diabetic peripheral neuropathy is the main form of neuropathy in diabetic neuropathy in 75% of the patients.The incidence rate increases with the prolonged duration of illness.Diabetic peripheral neuropathy seriously affects the quality of life of diabetic patients.Cardiac autonomic nerve is divided into sympathetic nerve and parasympathetic nerve.The occurrence of cardiac autonomic neuropathy is related to autonomic nervous system disease.Previous laboratory studies have shown that cervical sympathetic ganglion is involved in cardiac autonomic neuropathy,stellate ganglion(SG)belongs to cervical sympathetic nerve ganglion.Glucokinase(GCK)is the key enzyme of glucose metabolism,which is distributed in the liver,pancreas,intestine,central nervous system and other organs of the human body.Studies have shown that glucokinase is involved in the pathological inflammatory process,but there is no report about glucokinase in stellate ganglion involved in diabetic cardiac autonomic neuropathy.Therefore,the purpose of this study is to explore the relationship between the upregulation of GCK in cervical sympathetic ganglia and diabetic cardiac sympathetic neuropathy and its role in cardiac sympathetic neuropathy in type 2 diabetic rats.Itch is a kind of unpleasant feeling that causes the body to scratch desire under harmful stimulation.According to the duration of itch,it can be divided into acute itch and chronic itch.Acute itch is the body’s defense response,while chronic itch can reduce the quality of life of patients,and may lead to ulcer,infection,depression,anxiety,and even suicide.Studies have shown that diabetes can be complicated with chronic itch.Itch is a common clinical symptom in patients with diabetes.Purinergic(P)receptors can be divided into P1 receptor and P2 receptor.Studies have shown that P2Y12 receptor is involved in peripheral nerve injury and sensory dysfunction.Itch may be a marker of peripheral neuropathy.At present,there is no research report on P2Y12 receptor involved in chronic itch.This project is the first to carry out the research on the pathological changes of P2Y12 receptor involved in type 2 diabetes mellitus complicated with chronic itch.Part oneObjective: To investigate the effect of P2X3 receptor on glucokinase related cardiac sympathetic neuropathy in stellate ganglion of type 2 diabetic rats.Methods: The rat model of type 2 diabetes mellitus(T2DM)was established by feeding with high fat and high sugar.The rats were randomly divided into four groups(n=10): control group,diabetes model group(DM group),diabetes model +GCK short hairpin RNA group(DM + GCK shRNA group)and diabetes model +negative control(NC)short hairpin RNA group(DM + NC shRNA group).(1)Blood glucose,heart rate,blood pressure and sympathetic discharge were detected.(2)Western blot(WB)and real-time quantitative Polymerase Chain Reaction(RT-q PCR)were used to detect the effect of GCK shRNA on the expression of GCK in stellate ganglion of diabetic rats.(3)The effects of GCK shRNA on the coexpression of GCK and Neu N(Neuron markers)in stellate ganglion and the co localization expression of GCK and tyrosine hydroxylase(TH,Sympathetic markers)in myocardial tissues were detected by double immunofluorescence staining.(4)Western blot,RT q PCR and immunofluorescence double labeling assay were used to detect the effect of GCK shRNA on the up-regulated expression of P2X3 receptor and the co localization expression of P2X3 and Neu N in SG of diabetic rats.(5)Whole cell patch clamp was used to record P2X3 agonist activated currents in isolated stellate ganglion neurons and cultured human embryonic kidney 293(HEK293)cells transfected with P2X3 plasmid.(6)Western blot was used to detect the effect of GCK shRNA on the expression of tumor necrosis factor α(TNF-α)in SG of diabetic rats.Results: After GCK shRNA treatment,the up-regulated GCK expression in SG of T2 DM rats was decreased.GCK shRNA treatment can also improve blood pressure,sympathetic activity,heart rate and heart rate variability in T2 DM rats.In contrast,the expression of P2X3 and TNF-α were significantly decreased after GCK shRNA treatment.The ATP activation current of SG neurons transfected with GCK plasmid was higher than that of control neurons.In addition,compared with HEK293 cells transfected with P2X3 receptor plasmid alone,the current activated by P2X3 agonist in HEK293 cells co transfected with GCK and P2X3 receptor plasmid was significantly increased.After transfection with GCK shRNA plasmid,ATP activated currents in isolated neurons of stellate ganglion and HEK293 cells co transfected with GCK and P2X3 receptor plasmids were decreased.In T2 DM rats,downregulation of GCK attenuates the diabetic cardiac sympathetic effect mediated by upregulation of GCK involving P2X3 receptor.Conclusion:The upregulated GCK in SG participates in the pathological process of diabetic cardiac sympathetic neuropathy,inflammation,and cardiac dysfunction.GCK in SG may cooperate with P2X3 receptor to induce cardiac sympathetic neuropathy in T2 DM rats.Knockdown of GCK in SG of DM rats by shRNA treatment can decrease the expression of GCK,reduce neuron sensing to high glucose and inflammatory mediators,and consequently improve the pathological changes of diabetic cardiac sympathetic neuropathy and cardiac dysfunction mediated by upregulated P2X3 receptor.GCK in SG neurons may serve as a new target for the treatment of T2 DM.Part twoObjective: The mouse model of type 2 diabetes mellitus was used to investigate the molecular mechanism of type 2 diabetes mellitus complicated with chronic itching mediated by P2Y12 receptor.Methods: The model of type 2 diabetes mellitus in mice with chronic itch was established.The mice were randomly divided into 6 groups: control group,diabetes mellitus chronic itch(DMI)group,DMI + P2Y12 shRNA group,DMI + NC shRNA group,DMI +Ticagrelor group,DMI + DMSO group.(1)The changes of thermal hyperalgesia,cold hyperalgesia,spontaneous itch and sciatic nerve conduction velocity were detected.(2)The content of reactive oxygen species(ROS)in dorsal root ganglion(DRG)was detected by chemical fluorescence.(3)The expression of P2Y12 receptor in DRG was detected by Western blot and RT-q PCR.(4)The co expression of P2Y12 and glial fibrillary acidic protein(GFAP)in DRG was detected by immunofluorescence double labeling assay.(5)The expression of NOD-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)/Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain(ASC)/interleukin-1β(IL-1β)/IL-18 in DRG was detected by Western blot and q PCR.(6)The levels of IL-1β and IL-18 in serum were detected by enzyme linked immunosorbent assay(ELISA).Results: The results showed that:(1)Compared with the control group,the thermal hyperalgesia and cold hyperalgesia of mice in DMI group was significantly increased,and the behaviors of thermal hyperalgesia and cold hyperalgesia were alleviated after treatment with P2Y12 shRNA or P2Y12 antagonist Ticagrelor.Compared with DMI group,there was no significant change in DMI + NC shRNA group or DMI + DMSO group.(2)The results showed that the spontaneous itch behaviors of mice in DMI group were significantly improved after treatment with P2Y12 shRNA or P2Y12 antagonist Ticagrelor.(3)Compared with the control group,the sciatic nerve conduction velocity in DMI group was significantly decreased(p<0.01),while after treatment with P2Y12 shRNA and P2Y12 antagonist ticagrelor in the model group,the sciatic nerve conduction velocity was significantly increased(p<0.01).There was no significant difference in DMI + NC shRNA group or DMI +DMSO group compared with DMI group(p>0.05).(4)The results of ROS detection showed that the fluorescence value of DMI group was much higher than that of control group(p<0.01).Compared with DMI group,the fluorescence value in DMI +P2Y12 shRNA group or DMI + ticagrelor group was significantly decreased(p<0.01),but there was no significant difference in DMI + NC shRNA group or DMI+ DMSO group compared with DMI group(p>0.05).(5)Western blot and q PCR results showed that the P2Y12 m RNA and protein levels in DMI group were significantly higher than those in control group(p<0.01).After treating the model group mice with P2Y12 shRNA or Ticagrelor,the m RNA and protein levels of P2Y12 were significantly decreased(p<0.01,p<0.05).After NC shRNA injection and DMSO treatment in DMI mice,there were no significant changes in P2Y12 m RNA and protein levels compared with untreated DMI group(p>0.05).(6)Immunofluorescence double labeling showed that P2Y12 receptor co expressed with GFAP,a marker of satellite glial cells(SGCs),suggesting that P2Y12 receptor mainly existed in SGCs.Moreover,the co-expression of P2Y12 receptor and GFAP was increased in the DMI group compared with the control group(p<0.05).The up-regulated co expression of P2Y12 receptor and GFAP in the DMI group was decreased by P2Y12 shRNA or P2Y12 antagonist ticagrelor treatment(p < 0.01).However,the co-expression of P2Y12 receptor and GFAP in NC shRNA or DMSO treatment group had no significant change compared with untreated DMI group(p >0.05).(7)The up regulation of NLRP3/caspase-1/ASC/IL-1β/IL-18 in protein and m RNA levels was decreased by P2Y12 shRNA or P2Y12 antagonist ticagrelor treatment.(8)The levels of IL-1β and IL-18 in serum of diabetic mice with chronic itch were significantly higher than those in control group(p<0.01).The serum levels of IL-1β and IL-18 in DMI + P2Y12 shRNA group or DMI + ticagrelor group were significantly lower than those in untreated DMI group(p<0.01).Compared with DMI group,there was no significant difference in serum levels of IL-1β and IL-18 in DMI + NC shRNA group or DMI + DMSO group(p > 0.05).Conclusion: P2Y12 receptor can induce the increase of ROS content in vivo after the activation of satellite glial cells,and then NLRP3 inflammasome activation,release increase of inflammatory cytokines,abnormal excitation of DRG neurons,and damage of peripheral nerve,which leads to chronic itch.P2Y12 receptor shRNA or P2Y12 antagonist ticagrelor treatment can inhibit these pathological changes,thus improving itching symptoms.