Study In The Effects Of Drugs On P2 Receptors In Dorsal Root Ganglia Mediated HIV Gp120 Plus Antiretroviral Therapy Associated Neuropathic Pain

Chapter 1 IntroductionInfection of Human immunodeficiency virus?HIV?and application of highly active antiretroviral therapy?HAART?often result in neuropathic pain in patients.Some research data show that HIV-1 envelope glycoprotein 120?gp120?and the antiretroviral drugs of nucleoside analogue reverse transcriptase inhibitor,such as 2?,3?-dideoxycytidine?ddC?are the main causing.Peripheral neuropathy can cause more release of adenosine triphosphate?ATP?which acts on the purinergic receptors expressed in neurons and satellite glial cells?SGCs?of the dorsal root ganglion?DRG?.Activation of purinergic receptors can activate the pathological interaction of neurons and SGCs by cascade processes,resulting the abnormal excitation of DRG neurons,which plays a critical role in forming and maintanence of neropathic pain.We hypothesize that P₂X₃ ? P₂X₇ ? P₂Y₁2 receptors are involved in the pathological interaction of neurons and SGCs,and mediate the neropathic pain induced by gp120 and dd C.In this study,we observe the effects of the purinergic receptor short hairpin RNA?sh RNA?and andrographlide?a traditional Chinese medicine monomer?acting on the P₂X₃?P₂X₇?P₂Y₁2 receptors.Western blotting,quantitative real-time polymerase chain reaction?q PCR?,immunohistochemistry,double labled immunofluorescence,bioinformatics prediction technology,whole cell patch clamp,calcium image ect were used in this study.We try to explore the concentration of ATP release,the expression levels of immflamation cytokines and P₂X₃?P₂X₇?P₂Y₁2 receptors,as well as the activation of the downstream signal pathway that may be involved in the pathological process of the interaction between neurons and SGCs.Our results were good for explaining the mechanism of the HIV and HAART induced neuropathic pain by multi-use of cell and animal experiments.Chapter 2 P2X₃ receptor mediate neuropathic pain induced by gp120Objective: This study aims to explore the role of the P2X₃ receptor in gp120-induced neuropathic pain and its possible mechanism.Methods: Neuropathic pain animal model was created by wrapping sciatic nerve of the rat with a piece of regenerated cellulose which was soaked in HIV gp120 solution.Neuropathic pain behavors were measured by testing the mechanical hyperalgesia and thermal hyperalgesia;Quantitative real-time PCR?q PCR?,western blotting and immunohistochemistry were used to assay the expression levels of the P2X₃ receptor and the extracellular regulated protein kinase 1/2?ERK 1/2?;ATPlite 1 step assay was used to test the concentration of the ATP release,whole cell patch clamp was used to test the inhibitory effect of A317491 on ?,?me-ATP-induced currents in DRG neurons.Results: Our data showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group.The expression levels of the P2X₃ m RNA and protein in gp120-treated rats were higher than those in the sham group.The P2X₃ antagonist A317491 decreased mechanical and thermal hyperalgesia and reduced the up-regulated expression of P2X₃ m RNA and protein in gp120 treatment rats,as well as the ERK1/2 phosphorylation.In addition,P2X₃ agonist ?,?-methylene ATP??,?-me ATP?-activated currents in gp120 treatment DRG neurons were higher than those in control neurons,which decreased by A317491.Molecular docking data showed that A317491 may interact with the gp120 protein.Conclusion: The inhibition of the P2X₃ receptor in rat DRG neurons relieved gp120-induced mechanical and thermal hyperalgesia.Chapter 3 P2Y₁2 receptor mediate neuropathic pain induced by gp120 and dd CObjective: This study aims to explore the role of the P2Y₁2 receptor in gp120 combined with ddC-induced neuropathic pain and its possible mechanism.Methods: A rat model of gp120+dd C-induced neuropathic pain was used inthis study.Neuropathic pain behavor was evaluated by the mechanical hyperalgesia and thermal hyperalgesia.QPCR and western blotting were used to test the expression levels of the P2Y₁2 receptor,the p38 MAPK,IL-1?,and TNF-? protein.Calcium image was used to measure the [Ca²+]i of primary cultured DRG SGCs.Double-labeling immunofluorescence was used to test the co-expression of P2Y₁2 receptor and glial fibrillary acidic protein?GFAP?in DRG.Results: When peripheral nerve exposured to HIV-gp120+dd C,mechanical and thermal hyperalgesia in gp120+dd C-treated model rats were increased.The gp120+dd C treatment increased the expression of P2Y₁2 receptor m RNA and protein in DRG SGCs.In primary cultured DRG SGCs treated with gp120+dd C,the levels of [Ca²+]i activated by the P2Y₁2 receptor agonist 2-?Methylthio?adenosine 5'-diphosphate trisodium salt?2-Me SADP?were significantly increased.P2Y₁2 receptor sh RNA treatment inhibited 2-Me SADP-induced [Ca²+]i in primary cultured DRG SGCs treated with gp120+dd C.Intrathecal treatment with a sh RNA against P2Y₁2 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines,decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats.Conclusion: The P2Y₁2 receptor was involved gp120+ddC induced neuropathic pain,downregulating the P2Y₁2 receptor inhibited the releasing of proimflamatory cytokines,blocking its downstream signal pathway.Reducing the activation of SGCs and the changing of the [Ca²+]i improved the pathological interaction between the DRG neurons and SGCs,then resulted in relieving mechanical and thermalhyperalgesia in gp120+dd C-treated rats.Chapter 4 Andrographolide inhibits P2X₇ receptor mediated neuropathic pain induced by gp120 and dd CObjective: In this study,we investigated whether andrographolide?Andro?can alleviate neuropathic pain induced by HIV gp120 plus dd C treatment and its possiblemechanism.Methods: The paw withdrawal threshold and the paw withdrawal latency were observed to assess pain behaviors in all groups of the rats.Experimental animals were randomly divided into control group,control combined with Andro treatment group,sham group,gp120 combined with dd C treatment group,gp120 plus dd C combined with A438079 treatment group,and gp120 plus dd C combined with Andro treatment group.Andro was by intrathecally injected at a dose of 25 ?g/20 ?l.The protein expression levels of the P2X₇ receptor,tumor necrosis factor-?-receptor?TNF-?-R?,interleukin-1??IL-1??,IL-10,phospho-extracellular regulated protein kinases?ERK??p-ERK?in the L4-L6 dorsal root ganglia?DRG?were measured by western blotting.Real-time quantitative polymerase chain reaction was used to test the m RNA expression level of the P2X₇ receptor.Double-labeling immunofluorescence was used to identify the co-localization of the P2X₇ receptor with glial fibrillary acidic protein?GFAP?in DRG.Molecular docking was performed to identify whether the Andro interacted perfectly with the rat P2X₇?r P2X₇?receptor.Results: Andro attenuated the mechanical and thermal hyperalgesia in gp120+dd C-treated rats and down-regulated the P2X₇ receptor m RNA and protein expression in the L4-L6 DRGs of gp120+dd C-treated rats.Additionally,Andro simultaneously decreased the expression of TNF-?-R and IL-1? protein,increased the expression of IL-10 protein in L4-L6 DRGs,and inhibited the activation of ERK signaling pathways.Moreover,Andro decreased the co-expression of GFAP and the P2X₇ receptor in the SGCs of L4-L6 DRG.Conclusion: Andro decreased the hyperalgesia induced by gp120 plus dd C.