Effect Of Protein Disulfide Isomerase(PDI) In DRG On Murine Pain Behaviors

Protein disulfide isomerase（PDI）is a mercapto oxidordeuctase,belonging to the superfamily of thioredoxins.The structure of PDI mainly includes two enzyme catalytic domains a and a’,which contain the active sequence of CGHC,two substrate binding domains b and b’,and endoplasmic reticulum residence signal KDEL at the C terminal.PDI mainly exists in the endoplasmic reticulum,is an oxidoreductase required for protein folding.Changes in PDI expression or enzyme activity are associated with a range of diseases,such as neurodegenerative diseases and cardiovascular diseases.In addition to being a soluble oxidoreductase in the endoplasmic reticulum,PDI also exists outside the cell.PDI can be secreted or transported to the cell surface.PDI on the cell membrane is involved in regulating many important physiological processes,including platelet activation,thrombosis,T cell migration and osteoma migration.Pain is an unpleasant sensation caused by peripheral synaptic terminals of somatosensory neurons receiving various noxious stimuli（temperature,mechanical and chemical stimuli）and converting them into nerve impulses to transmit to the central nervous system.Pain can be used as a warning of injury to the body,causing a series of defensive reactions.Somatosensory neurons are located in dorsal root ganglion（DRG）,and tremendous nerve endings innervate various surrounding tissues,including skin,joints,respiratory tract,etc.DRG neurons,as primary somatosensory afferent neurons,play an extremely important role in pain signal transduction.Some studies have found that DRG neurons not only have the function of pain signal formation and transmission,but also have the function of pain signal regulation.In addition,it has been found that the expression of PDI in DRG and spinal cord increased in pain models.The symptoms of pain can be alleviated by PDI inhibitors through increasing the pain threshold in mice.Our previous experiments also found that specific knockout of PDI from Nav1.8⁺DRG neurons could alleviate acute inflammatory pain in mice induced by bradykinin（BK）.In order to further confirm the relationship between PDI and pain,this paper aims to explore the expression and distribution of PDI in DRG tissue,and the change of PDI protein expression in DRG in murine chronic pain,and to demonstrate the role of PDI in pathological pain.Part 1 Distribution of protein disulfide isomerase in mouse DRG and its effect on acute painObjective:To test the expression and distribution of PDI in DRG and primarily explore the effect of exogenous PDI on pain behavior of mice.Methods:（1）Frozen section and immunofluorescence techniques were used to observe the co-expression of PDI and DRG-related Markers such as IB4,CGRP and NF200.（2）ELISA kit was used to detect the secretion of PDI from DRG tissues.（3）Flow cytometry was used to detect the presence of PDI on the surface of DRG neurons.（4）Recombinant protein disulfide isomerase hPDI and mutant protein PDIoooo were prepared through E coli system.SDS-PAGE was used to detect the purity of recombinant protein.Di-E-GSSG was used as a substrate to detect the reductive activity of the recombinant proteins.（5）hPDI and PDIoooo were injected into the right hind paw of wild type mice,respectively.The withdrawal threshold to mechanical stimulation and latency to thermal stimulation of mice were measured at 0,1,3,5 and 24hours after injection.Thermal and mechanical thresholds were tested using Hargreaves and von Frey tests.Results:（1）PDI was shown co-labeled with DRG neurons markers IB4,CGRP,NF200 in certain extents via immunofluorescence measurement.Among them,NF200（neurofilament protein）is a marker labeling large diameter cells in DRG;CGRP（calcitonin gene-related peptide）is a molecular marker specifically expressed in peptide-like small neurons;IB4（plant lectin）is a molecular marker of non-peptide-like small neurons.These results suggest that PDI is expressed in both small and large DRG neurons.（2）ELISA assay uncovered the fact of PDI secretion from DRG tissue.Compared with the control group（normal DRG extracellular solution stimulation）,high K⁺and BK stimulation did not increase or decrease the secretion of PDI.（3）Flow cytometry experiment revealed that PDI exists on the surface of DRG neurons.High K⁺stimulation did not affect the amount of PDI on the surface and the proportion of PDI positive cells.（4）The preparation,purity test and enzymatic activity measurement of recombinant protein disulfide isomerase hPDI and PDIoooo.Recombinant hPDI and PDIoooo protein were prepared via E.coli amplification system with concentration of 7.50 mg/ml and 8.22 mg/ml respectively.The purities of hPDI and PDIoooo protein were detected as 90%and 85%through SDS-PAGE.Strong reductive activity of hPDI was detected by Di-E-GSSG assay,while the mutant protein PDIoooo was almost null activity.（5）The primarily study of effect of recombinant protein PDI on the pain behavior of mice.Compared with the control group,the thermal threshold of the mice significantly decreased in the hPDI injection group,especially at 3th and 5th hours after injection.While the mechanical threshold of hPDI injected mice did not change significantly compared with the control group,although there were significantly different between two groups at the 5th and 24th hours after injection.Conclusions:1.PDI protein expressed in both large and small DRG neurons;2.PDI can secret from DRG neurons and exist on the cell surface;3.Exogenous administration of hPDI reduce pain thresholds in mice,particularly induce thermal hyperagesia.Part 2 Changes of PDI protein in DRG and its effect on chronic painObjective:To study the changes of PDI protein in DRG and to explore its role in chronic inflammatory and neuropathic pain in mice.Methods:（1）CFA chronic inflammatory murine model was used to detect the changes of PDI protein in DRG.CFA（25μl）was injected to the right hind paw of wild type mice and the control mice were injected with saline（25μl）.The total protein was extracted from L3,L4 and L5 DRGs on the 3^rd,5^th and7^th days after injection to detect PDI expression using Western Blot.（2）CCI chronic neuropathic pain model was used to detect the changes of PDI protein in DRG.Sciatic nerve was exposed and four surgical sutures（chromic gut）were loosely ligated around the nerve,the sham group was operated in parallel without ligation of sciatic nerve.The total protein was extracted from L3,L4 and L5 DRGs on the 5^th,7^th and 10^th days after operation to detect PDI expression using Western Blot.（3）SNS-Cre,PDI^fl/fl mice and PDI^fl/fl mice were subjected to CFA injection.Thermal and mechanical thresholds were tested using Hargreaves and von Frey tests.Withdrawal latency and mechanical threshold of SNS-Cre,PDI^fl/fl mice（experimental group）and PDI^fl/fl mice（control group）were measured before and on the 1^st,3^rd,5^th,7^th,9^th,11^th,13^th and 15^th days after CFA injection.（4）SNS-Cre,PDI^fl/fl mice and PDI^fl/fl mice were subjected to CCI surgery.Thermal and mechanical thresholds were tested using Hargreaves and von Frey tests.Withdrawal latency and mechanical threshold of SNS-Cre,PDI^fl/fll/fl mice（experimental group）and PDI^fl/fl mice（control group）were measured before and on the 1^st,3^rd,5^th,7^th,10^th,and 14^th days after CCI surgery.（5）DNA extraction and genotyping of Advillin-CreERT2,PDI^fl/fl mice and PDI^fl/fl mice.2 mg Tamoxifen was intraperitoneally injected for four consecutive days at the age of 8 weeks of mice to knockdown PDI from DRG.The efficiency of knockdown was confirmed using Western Blot.（6）CFA（25μl）was injected into the right hind paw of the PDI^fl/fl mice and Advillin-CreERT2,PDI^fl/fl mice treated with Tamoxifen.Withdrawal latency and mechanical threshold of Advillin-CreERT2,PDI^fl/fl mice（experimental group）and PDI^fl/fl mice（control group）were measured before and at the 1^st,3^rd,5^th,7^th,10^th,and 14^th days after CFA injection using Hargreaves and Von Frey tests.Results:（1）The expression of PDI protein in DRG of wild type mice was significantly up-regulated after CFA injection.The results showed that,compared with control mice there were about 1.22±0.04,1.78±0.23 and1.79±0.14 fold increase of PDI expression in DRG on the 3^rd,5^th and 7^th days after CFA injection.（2）The expression of PDI protein in DRG of wild type mice was significantly up-regulated after CCI operation.The results showed that,compared with control mice there were about 1.52±0.04,1.32±0.07 and1.30±0.07 fold increase of PDI expression in DRG on the 5^th,7^th and 10^thh days after CCI operation.（3）Behavioral results of SNS-Cre,PDI^fl/fl mice in CFA model:Compared with PDI^fl/fl mice,SNS-Cre,PDI^fl/fl mice showed significantly longer withdrawal latency to heat stimulation on the 5^th,7^th,9^th,11^th and 13^th days after CFA injection.And the overall inter-group difference between SNS-Cre,PDI^fl/fl mice and PDI^fl/fl mice in thermal threshold was satistically significant.However,the mechanical threshold was significantly reduced only on the 13^thh day after CFA injection in SNS-Cre,PDI^fl/fl mice.The overall inter-group difference between the two groups in mechanical threshold did not reach statistical significance.（4）Behavioral results of SNS-Cre,PDI^fl/fl mice in CCI model:Compared with PDI^fl/fl mice,SNS-Cre,PDI^fl/fl mice showed significantly longer withdrawal latency to heat stimulation on the 7^th,10^th days after CCI surgery.And the overall inter-group difference between SNS-Cre,PDI^fl/fl mice and PDI^fl/fl mice in thermal threshold was statistically significant.There were no difference in mechanical thresholds on all the tested days between SNS-Cre,PDI^fl/fl mice and PDI^fl/fl mice.The overall inter-group difference between the two groups in mechanical threshold did not reach statistical significance.（5）Preparing and genotyping Advillin-CreERT2,PDI^fl/fl transgenic mice:Advillin-CreERT2 transgenic mice were mated with PDI^fl/+transgenic mice to obtain Advillin-CreERT2,PDI^fl/+heterozygous mice,and then mated with PDI^fl/+mice to obtain Advillin-CreERT2,PDI^fl/fl mice.Two weeks after birth,the toes of the mice were cut offand the genomic DNA was extracted.The DNA bands were detected by PCR amplification and gel electrophoresis.The target band of Cre gene is about 180 bp,the WT band of PDI is about 480 bp,and the Flox band of PDI is about 550 bp.Efficiency of PDI knockout in DRG tissues:Advillin-CreERT2,PDI^fl/fll/fl mice were injected intraperitoneally with Tamoxifen for four consecutive days at the age of 8 weeks to induce PDI knockdown.The total protein of DRG tissue were extracted from Advillin-CreERT2,PDI^fl/fl mice and PDI^fl/fl mice,then subjected to western blot.The results showed that compared with PDI^fl/fll/fl mice,the amount of PDI protein in DRG tissue of Advillin-CreERT2 and PDI^fl/fl mice decreased slightly.（6）Behavioral results of Advillin-CreERT2 and PDI^fl/fl mice in CFA model:In CFA chronic inflammatory pain model,withdrawal latency of Advillin-CreERT2 and PDI^fl/fl mice tended to increase,but there was no significant difference compared with PDI^fl/fl mice.No significant difference in mechanical threshold was observed between the two groups.Conclusions:1.The expression of PDI protein in DRG tissue was increased in CFA chronic inflammatory pain model and CCI chronic neuropathic pain model.2.Specific knockout of PDI from nociceptive DRG neurons could alleviate the chronic inflammatory pain and neuropathic pain symptoms in mice.