Effects And Mechanisms Of Conditional PDI Knockout In DRG Nociceptor Neurons On Mice Pain Behaviors

Protein disulfide isomerases?PDIs?family belongs to thioredox superfamily and shares the similar active structures CXXC,PDIs can oxidate,reduce and isomerate disulfide bond and have the function of molecular chaperon.At present,more than 20 members of PDIs family have been identified,amongst which PDI is the prototype one.We found in the preliminary study that PDI is highly expressed in neurons of dorsal root ganglia?DRG?.DRG neurons are the primary somatosensory neurons,and DRG is an important location for the pain signal initiation and propagation.It has been reported that protein disulfide isomerases PDIA1,PDIA3?ERp57?,PDIA6 are upregulated significantly in CFA?complete Freund's adjuvant?inflammatory chronic pain model;futhermore,intraperitoneal injection PDI antagonist PACMA31 tempers the CFA-induced mechanical pain.These evidences suggest PDI in DRG may serve important role in pain.To provide experimental evidence for this hypothesis,we first used Cre-loxP recombinase system to generate nociceptors-specific PDI knockout mice by knocking out PDI from Nav1.8 voltage-dependent sodium channel positive DRG neurons.Then we demonstrated the role of PDI by testing the basic threshold of mechanical and thermal pain,acute inflammatory pain?caused by injection of bradykinin?BK?in the plantar of right hind paw?and chronic inflammatory pain?caused by injection of CFA in the plantar of right hind paw?behaviors in conditional kochout mice.It is known that ion channels are the basis of DRG neurons excitation and initiation of pain signals.Effects of PDI on ion channels have been sporadically reported.For example,it is reported that PDI increases the activity of Gardos channel?Potassium intermediate/small conductance calcium-activated channel,subfamily N,member 4,KCNN4?in erythrocytes.PDI potentiates NMDA receptor's activity by reducing its disulfide bond in hippocampus neurons of mice,which leads to hyperexcitability of neurons and seizure.So far the effects of PDI on excitability of DRG neurons have not been demonstrated.In this study,we tested the effects of recombinant protein disulfide isomerase hPDI on the excitability of DRG neurons by patch clamp technology,and then we studied the molecular mechanisms of PDI actions by testing the effects of PDI on M-type K⁺channel and T-type Ca²⁺channels.Part 1 Effects of Conditional PDI knockout in nociceptors on painObjective:Using Cre-loxP recombination system to knock out PDI in nociceptors to study the roles of PDI in primary nociceptive neurons during pain sensation.Methods:?1?Mice with selective deletion of PDI in Nav1.8-expressing DRG neurons were generated by mating SNS-Cre mice[mice expressing Cre recombinase under regulatory element of mouse Nav1.8 gene?Scn10a?]with PDI-loxP mice[exon 1 and exon2 of Pdi floxed by loxP].Efficiency of PDI knockout was validated by genotyping and immunofluorescence imaging.?2?Basic thresholds of mechanical and thermal pain were measured using Von Frey filament test and Hargreaves test,respectively.?3?Acute inflammatory pain behavior was induced by injection of BK?200?M,25?l?into right hind paw of mice.The nocifensive behaviors were recorded with video cameras.?4?Chronic pain was induced by injection of CFA?20?l?into right hind paw of mice.The behaviors of hyperalgesia and allodynia to mechanical and thermal stimulis were tested for 14 days after CFA injection.?5?Statistic methods:Mann-Whitney U test,t'test and one-way reapeated measures ANOVA analysis were used.Results:?1?Generation and genotyping of nociceptors-specific knockout PDI mice:SNS-Cre,PDI^fl/+heterozygus mice were generated by crossing SNS-Cre mice with PDI^fl/+ll/+l mice.Then the SNS-Cre,PDI^fl/+heterozygus were crossed with PDI^fl/+to get SNS-Cre,PDI^fl/fll/fl mice,which were the nociceptors-specific PDI knockout mice.About ten days after birth,all the mice were marked by toe cutting,and genomic DNA was extracted from tail of mice.Transgenic mice identification was done using PCR technique followed by gel electrophoresis.The target band of Cre was about 350 bp,while the band of PDI in WT mice was about 450 bp and Flox?flanked by loxP?band of PDI was about 550 bp.?2?Efficiency of PDI conditional knockout in DRG:Frozen DRG sections were prepared from SNS-Cre,PDI^fl/fll/fl and PDI^fl/fll/fl mice,followed by immunofluorescence imaging to observe the expression of PDI.Rich expression of PDI was found in DRG tissue of PDI^fl/fll/fl mice and the expression was markedly decreased in SNS-Cre,PDI^fl/fll/fl mice.?3?Pain behavioral tests:Under basic conditions,the mechanical pain thresholds of SNS-Cre,PDI^fl/fll/fl mice were significantly increased?P<0.01?compared with that of PDI^fl/fl mice,while the thermal thresholds were not changed.In acute inflammatory pain model,within 15 min injection of BK in right hind paw of mice,the nocifensive behaviors?total licking time and numbers of licking?were significantly decreased?P<0.05?in SNS-Cre,PDI^fl/fll/fl mice compared with PDI^fl/fll/fl mice.However,in chronic inflammatory pain model with injection of CFA in hind paw of mice,there were no differences in response to mechanical and thermal stimulis between SNS-Cre,PDI^fl/fl and PDI^fl/fll/fl mice.Conclusions:These results suggest PDI plays an important role in origination and propagation of pain signal.Conditional PDI knockout from nociceptive DRG neurons significantly reduces the sensitivity to mechanical stimulation and alleviates the symptoms of acute pain in mice.Part 2 Effects of purified active protein hPDI on the excitability of DRG neuronObjective:To observe the effects of PDI on the excitability,M-type K⁺currents and T-type Ca²⁺currents of DRG neurons;thus to explore the molecular mechanisms of PDI on DRG neurons.Methods:?1?Purified recombinant human protein disulfide isomerase?hPDI?was obtained in vitro from expressed hPDI in E.coli expression.Enzyme activity of purified hPDI was assayed by measuring its reductase activity against substrate Di-E-GSSG.?2?Patch-clamp technology was used to observe the effects of PDI on the excitability,M-type K⁺currents and T-type Ca²⁺currents of DRG neutrons.?3?Statistic methods:Paired t-test and two-sample t test were used.Results:?1?Preparation and activity assay of purified hPDI:Four batches of hPDI with different concentrations were purified:C1=3.5 mg/ml,C2=2.1 mg/ml,C3=1.9 mg/ml,C4=2.7 mg/ml.The disulfide bone in Di-E-GSSG can be reduced by PDI?100 nM?thus to produce EGSH which could be measured by its fluorescence activity.The test results showed the second and fourth batches of protein have similar and high reduction activity and were used in following electrophysiology experiments;the reductivity of the first and third batch of purified proteins were relatively low.?2?Effects of hPDI on the excitability of DRG neurons:Application of hPDI?100 nM?in extracellular depolarized membrane potential significantly?P<0.01?and the excitability of DRG neurons were significantly increased?P<0.01?.We also tested the effects of inactive PDI,PDIoooo?100 nM?,in which the two active sites CGHC of protein were both mutated to SGHS.Application of PDIoooo?100 nM?only slightly depolarized the membrane potential.And the number of induced action potential were also slightly increased by application of PDIoooo?100 nM?.Howere,both of the effects from PDIoooo were significantly lower than that of hPDI?P<0.05?.?3?Effects of hPDI?100 nM?on M-type K⁺currents and T-type Ca²⁺currents:hPDI?100 nM?had no significant effects on these two channel currents.Conclusions:The results suggest PDI increases the excitability of DRG neuron,and this effect was not like through modulation of M-type K⁺currents or T-type Ca²⁺currents.The two active sites CGHC of PDI are crucial for PDI action.