The Role Of CBS/P2X Signaling Pathways In Painful Diabetic Neuropathy

Objective:Painful diabetic neuropathy is one of the most common types of diabetes complications. The underlying molecular mechanism of diabetic mechanical allodynia(DMA)has not fully been elucidated,the change of ion channel may be involved in its development.As ionotropic ligand-gated ion channel receptors,the purinergic P2 X receptors are activated by the extracellularly released adenosine triphosphate(ATP),which increases the excitability of primary afferent neuronal and activates pain pathways,that eventually results in hyperalgesia. Hydrogen sulfide(H2S)was one of very important peripheral conditioning substance in the process of pain. The regulation of pain was related to the regulation of voltage gated calcium channels.Cystathionine-beta-synthase was the key enzyme when nervous tissue produced endogenous H2 S,thus which played an important role in the process of pain.Our study observed temporal and spatial changes of P2X3 receptors m RNA and protein levels in diabetic mechanical allodynia model rats’ dorsal root ganglion at different time point. At the same time,we also observed temporal and spatial changes of P2X4 receptors m RNA levels and CBS protein levels at different time point.This study aimed to uncover the molecular mechanism of diabetic mechanical allodynia and provided new method and experimental basis for clinical treatment.Methods:70 healthy male Sprague-Dawley rats were randomly divided into normal group(n =10) and model group(n = 60). Diabetic model was induced by a single intraperitoneal injection of streptozotocin(STZ; 60 mg/kg, i.p.). The normal group was injected with isodose citric acid citrate sodium buffer solution. Three days later,only rats with blood glucose concentration >16.7 mmol/L were further used in the study.Seven days later, The painful threshold of diabetic model rats decreased by 50% than before STZ injection were DMA rats and further used in the study.Then the DMA rats were randomly divided into 7days, 14 days, 21 days and 28 days Group. Rat tail vein blood glucose, body weight and diabetic mechanical allodynia were determined before, after 3 days,7 days,14 days,21days,28 days STZ or NS injection. Quantitative real-time PCR was used to measure the m RNA level of P2X3 receptor and P2X4 receptor in the rats DRG.Western blot was performed to detect the protein level of P2X3 receptor and CBS in the rats DRG.Results:Rats in the diabetic model group showed conspicuous symptoms like yellow hair,polyphagia, polydipsia, polyuria and weight loss.,Approximately 50% of DM rats displayed significantly decrease of paw withdrawal threshold(PWT) 7 days after STZ injection. The lowest point of PWT appeared 21 days and 28 days after STZ administration.Blood glucose were significant higher than those of control group.Compared with the control group, RT-PCR experiments revealed expressions of P2X3 and P2X4 were significant higher on 14 days in the DMA group.Western blotting results showed that the temporal changes of P2X3 was not in accordance with their m RNA changes, while expressions of P2X3 was still higher than the control group on 21 days and 28 days. Western blot analysis showed that expressions of CBS was significant higher on 14 days,21 days and 28 days compared with the control group.Conclusions:1. The major features described in diabetes(polyphagia, polydipsia, polyuria and weight loss) are conspicuous by a single intraperitoneal injection of streptozotocin.At the same time,STZ also could make rats produce mechanical pain threshold and hyperalgesia.2. Expressions of P2X3, P2X4 and CBS were significant higher in the DMA group,which showed they contribute to mechanical hyperalgesia in STZ-induced diabetic rats and may become painful diabetic neuropathy treatment of potential targets.