| Background:Diabetic neuropathic pain(DNP),one of the most common complications of diabetes mellitus,is often characterized by spontaneous pain,hyperalgesia and allodynia neuropathic pain.Approximately 50%of diabetic patients suffer from DNP,and the neuropathic lesion happens in the early stage of diabetes..The development and maintenance of diabetic neuropathy are extremely complex and intertwined,and the exact mechanism remains unclear.Diabetic neuropathic pain induced mental anguish and often brought economic burden to patients,therefore the studies of developing DNP preventive and therapeutic targets are imperative.Dorsal root ganglia(DRG),the primary sensory neuron,accept nerve impulse from peripheral receptor activated by nociceptive stimuli and transmit the signal to the center nervous system.Noxious stimulation cause the cell damage and stress,which further induce the release of abundant inflammatory substances,such as adenosine triphosphate(ATP),from damaged or stressed cells and nerve terminals.ATP can activate P2X receptors,which is involved in the nociceptive signaling transmission of neuropathic pain.The primary sensory ganglia,particularly in DRG,contain the cell bodies of sensory neurons and satellite glial cells(SGCs)closely surrounding neurons.Nerve injury induces abnormal thickening of SGC,upregulated expression of glial fibrillary acidic protein(GFAP),enhancement of coupling between satellite glial cells mediated by gap junction(GJ).Inhibitor of gap junction can reduce the abnormal discharge of primary sensory neurons in animal model of pain.Research showed that the sensitivity of SGCs to ATP in primary sensory ganglia was increased after nerve injury.Because of the abnormal changes of the gap junction,the cell to cell communication in primary sensory ganglia mediated by ATP is enhanced,thereby strengthening the neuron discharge and causing pain.Diabetic rat models were used to study on prevention and treatment of neuropathic pain mediated by P2X receptors in DRG and the underlying mechanisms.Studies have shown that inflammatory thermal pain sensitivity and mechanical pain sensitivity in P2X7 receptor knockout mice were lower than those in wild type mice.Antagonist of P2X7 receptor alleviates pain behaviors of neuropathic pain rats.These results suggest that P2X7 receptor may involved in diabetic neuropathic pain.There are two main changes in primary sensory ganglia after injury,which are the increasement of GJs mediated cell coupling and the enhancement of the ATP sensitivity to P2X receptors in SGCs and neurons.SGCs in DRG do express P2X7 receptor.Because the abnormal changes of GJs and ATP mediated communication within the primary sensory ganglion cells,the neuronal discharge is enhanced and further causes pain.The first section of this study will explore the role of P2X7 receptor in SGCs of DRG and GJ protein(connexin 43)in DNP rat model.Neuropathic pain increased the expression of P2X4 receptor in activated microglia of spinal cord dorsal horn,which is associated with pain behaviors in animals.Downregulating the expression of P2X4 receptor protein in neuropathic pain rats can alleviate pain behaviors.P2X4 receptor is expressed in DRG SGCs.These results indicate that P2X4 receptor is possible involved in the pathological changes of DNP.Inflammation and oxidation are related to the pathological processes of DNP.Hesperidin performs anti-inflammatory and antioxidant effects,which suggests it may affect DNP.The second section of this study will observe the effect of hesperidin on DNP mediated by P2X4 receptor in DRG SGCs and its possible mechanisms.Ras gene belongs to proto-oncogene encoding intracellular signaling proteins.K-ras,a member of the ras gene family,encodes K-ras proteins.K-ras plays an important role in multiple cell signaling pathways as a molecular switch.K-ras is involved in the transmission of nociceptive information.The third section of this study will investigate the effects of K-ras small interference RNA(siRNA)on DNP mediated by P2X7 receptor in DRG,which will potentially provide a new target for the preventive and therapeutic strategy of DNP.It has shown that P2X3 receptor in DRG is involved in the transmission of pain.Puerarin can inhibit the transmission of nociceptive information mediated by P2X3 receptor in DRG neurons,and the molecular mechanism is still unkown.The fourth section of this study will observe the effects of puerarin on ATP activated currents in wild type-or mutant-P2X3 plasimid transfected-HEK293 cells.We can analysis the site of P2X3 receptor that puerarin interacting with.The results will provide further understanding of the molecular mechanisms that puerarin acting on P2X3 receptor.Objective:1.To investigate the role of P2X7 receptor in SGCs and GJ protein(connexin 43)in DRG in DNP rats.2.To examine the effects of hesperidin on DNP mediated by P2X4 receptor in DRG SGCs and the possible mechanisms.3.To explore the effects of K-ras siRNA on DNP mediated by P2X7 receptor in DRG were studied by using DNP rats.It will provide a new target for the preventive and therapeutic strategy of DNP.4.To explore the binding sites of P2X3 receptor that puerarin interact with and determine the molecular mechanisms of puerarin inhibiting P2X3 receptor activation.Method:1.Blood glucose levels in diabetic rat models were measured to determine whether or not the model is successfully established.Diabetic rats were injected with suramin(an antagonist of P2X receptor)and CBX(GJ protein blocker),then tested the mechanical pain threshold and thermal pain threshold in diabetic rats.The expression of GFAP(SGC marker)and the co-localization with P2X7 receptor in DRG SGCs were detected by double immunofluorescence staining.The expression levels of P2X7 receptor in DRG were detected by RT-PCR and Western blot.The expression levels of Cx43 were detected by Western blot.We observed the pathologic role of P2X7 receptor and GJ protein in DRG SGCs of DNP rats.2.Rats were fed with high sugar and high fat diet in combination with small dose of STZ injection to induce type 2 diabetic rat model.Diabetic rats were treated with hesperidin every day.Mechanical pain threshold and thermal pain threshold in all rats were tested.The expression levels of P2X4 receptor in DRG were detected by RT-PCR,immunohistochemistry and Western blot.The expression values of TNF-R1 and ERK protein were detected by Western blot.The effects of hesperidin on ATP-activated currents in HEK293 cells transfected with P2X4 recombinant plasmid were observed by whole cell patch clamp technique.3.Interaction between RNA(K-ras)and protein(P2X7)can be predicted by application of bioinformatics technology.According to the RNA and the secondary structure of protein,hydrogen bonds,and intermolecular forces,the interaction between K-ras and P2X7 was assessed.After intravenous injection of K-ras siRNA in diabetic rats,mechanical pain threshold and thermal pain threshold in rats were tested.The expression levels of P2X7 receptor in DRG were detected by RT-PCR,immunofluorescence and Western blot.Immunofluorescence and Western blot were performed to detect the expression of GFAP and TNF-a in rat DRG.ATP-activated currents in DRG neurons of diabetic rats and control rats were observed by whole cell patch clamp technique,and the difference between the two groups was analyzed.ATP-activated currents in HEK293 cells transfected with pEGFP-P2X7 only or transfected P2X7 together with K-ras siRNA were recorded and compared.4.The binding sites of P2X3 receptor that puerarin interact with were determined by homology modeling and molecular docking.The docking score of protein(P2X3 receptor)and ligand(puerarin)was analyzed by application of Ligand Poses process of Discovery Studio 3.5 software and chose the P2X3 mutation point according to the docking score.Binding sites of P2X3 receptor with puerarin was mutated by overlap extension PCR technique.The mutant of pEGFP-C1-P2X3 recombinant plasmid was constructed.The effects of puerarin on ATP-activated currents in HEK293 cells transfected with wild type or mutant P2X3 receptor were observed by whole cell patch clamp technique.Result:1.Fasting plasma glucose(FPG)and postprandial blood glucose(PBG)levels in diabetic rats were higher than those in control rats.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were significantly decreased in diabetic rats compared with control rats,while mechanical withdrawal threshold and thermal withdrawal threshold in diabetic rats treated with suramin and CBX rats at 7 d and 14 d were increased compared with those in daibetic rats.The expression values both P2X7 receptor and GFAP in DRG SGCs of diabetic rats were more than those in control rats,while the expression values in diabetic rats treated with suramin group and CBX group were both decreased compared with those in diabetic rats.The expression levels of GFAP protein,P2X7 mRNA and protein,Cx43 protein in DRG were increased in diabetic rats compared with control rats,while the expression levels were decreased in diabetic rats treated with suramin group and CBX group compared with diabetic rats.2.After feeding with high sugar and fat diet in combination with small dose STZ injection,FPG and PBG levels were higher in diabetic rats than those in control rats,which showed that type 2 diabetic models were successfully established.MWT and TWL in diabetic rats were significantly decreased compared with those in control rats,while they were increased in diabetic rats treated with hesperidin group compared with those in diabetic rat group.The expression levels of P2X4 receptor mRNA and protein,TNF-R1 protein,phosphorylation ERK protein(activation of ERK)in DRG were significantly increased in diabetic rats compared with control rats,while they were obviously reduced in diabetic rats treated with hesperidin group compared with diabetic rat group.Electrophysiology results showed that hesperidin significantly inhibited ATP-activated currents in HEK293 cells transfected with pEGFP-hP2X4 receptor.3.The catRAPID analysis showed that the interaction score of K-ras RNA and P2X7 receptor was relatively high.MWT and TWL were significantly decreased in diabetic rats compared with control rats,while they were significantly increased in diabetic rats treated with K-ras siRNA group compared with diabetic rat group.Sensory nerve conduction velocity(SCV)in diabetic rats was significantly decreased compared to control rats,while SCV was significantly increased in diabetic rats treated with K-ras siRNA group compared with diabetic rat group.The expression levels of P2X7 mRNA and protein in DRG were increased in diabetic rats compared with control rats,while they were decreased in diabetic rats treated with K-ras siRNA compared with diabetic rats.P2X7 receptor can co-locate with GFAP in DRG SGCs of diabetic rats.The expression values both P2X7 receptor and GFAP in DRG of diabetic rats were more than those in control rats,while they were decreased in diabetic rats after treated with K-ras siRNA.Electrophysiology results show that ATP-activated currents in DRG neurons of diabetic rats were significantly increased than those in control rats.ATP-activated currents in HEK293 cells transfected with pEGFP-P2X7 and K-ras siRNA were smaller than those in HEK293 cells only transfected with pEGFP-P2X7.4.Puerarin can bind well with human P2X3(hP2X3)protein located close to the ATP pockets and its surrounding residues,and formates hydrogen bonds with hP2X3 protein.Results of MOE score software concluded that puerarin can dock best with hP2X3 protein with the dock energy-6.1829 kcal/mol,which was greater than-3.81 kcal/mol between hP2X3 and ATP.We speculated that when puerarin binding to hP2X3 protein,it caused the restrictions of ATP binding with hP2X3 protein,which increased the concentration of free ATP,further leaded to inhibition of the activity of hP2X3 protein.Dock energy after point mutations of GLY130,VAL64,THR82,GLN85 on P2X3 receptor were tested respectively,we found that the mutation of VAL64 was very important in the docking results of protein-ligand.LibDockScores of P2X3 receptor docking with ATP or puerarin were 154.924 and 122.257 kcal/mol,respectively.It shows that VAL64 mutation can reduce the interaction of puerarin with P2X3 receptor,but had little effect on the direct interaction of ATP with P2X3 receptor.Electrophysiology results show that puerarin has obvious inhibitory effect on ATP-activated currents in HEK293 cells transfected with wild type P2X3 receptor,while the inhibition in HEK293 cells transfected with mutant P2X3 receptor was significantly decreased.Conclusion:1.After diabetic nerve injury,the expression levles of GFAP and GJ protein 43 in DRG were significantly increased.The expression values of P2X7 receptor in DRG SGCs were also increased significantly.Suramin(a P2X receptor inhibitor)suppressed over-expression of P2X7 receptor and inhibited the communication between neuron and SGC,and thereby reduced the discharge of DRG neurons and decreased the sensitivity of mechanical pain and thermal pain in diabetic rats.2.After diabetic nerve injury,the expression levels of P2X4 receptor in DRG were increased,which promoted the release of inflammatory cytokines from SGCs,increased the expression levels of TNF-Rl and phosphorylation ERK,and enhanced mechanical and thermal pain sensitivity of diabetic rats.Hesperidin inhibited the expression of P2X4 receptor in DRG,reduced the release of inflammatory cytokines mediated by abnormal activation of P2X4 receptor in SGC,reduced abnormal excitability of DRG neurons mediated by inflammatory cytokines,and decreased the sensitivity of mechanical pain and thermal pain in diabetic rats.3.P2X7 receptor in DRG plays an important role in DNP.K-ras siRNA treatment in type 2 diabetic rats can reduce the expression of P2X7 receptor and GFAP in DRG SGCs,reduce the release of inflammatory cytokines,thereby inhibit the hyper-excitability of DRG neurons,and relieve the pain behaviors of diabetic rats.4.VAL64ALA mutant site in P2X3 receptor was found by molecular docking method,and the result was verified by performation of cell transfection and whole cell patch clamp experiments.After point mutation of VAL64ALA site,the interaction between puerarin and P2X3 receptor was decreased,but it have no effect on the interaction between ATP and P2X3 receptor.Therefore,we speculate that VAL64ALA amino acid site is an important binding sites of P2X3 receptor that puerarin interact with P2X3 receptor.Puerarin can inhibit ATP-activated currents conducted by P2X3 receptor by biding with VAL64ALA amino acid residue. |
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