Stem Cells From Human Exfoliated Deciduous Teeth Relieve Trigeminal Neuralgia In Rats Via Paracrine Action On PD-1

Objective: To construct model of chronic constriction injury-infraorbital nerve(CCI-ION),and to observe the changes of mechanical pain threshold after local transplantation of SHEDs(Stem Cells from Human Exfoliated Deciduous Teeth)in CCI-ION rats.The effect of PD-1 blocking antibody nivolumab on pain relief in SHEDs transplantation rats after ligation was observed.To explore whether SHEDs can relieve pain by regulating the expression of TRPA1 and CGRP in the trigeminal ganglion(TG)via programmed Death protein 1(PD-1).To explore whether SHEDs can induce analgesic effect via paracrine action on PD-1 by co-culture system of LPS-pretreated sh-sy5y(human neuroblastoma cell line)and SHEDs.This study will provide experimental foundation and novel target for the mechanisms of stem cell therapy of trigeminal neuralgia.Methods: 1.Primary SHEDs were isolated and cultured,and the surface markers of mesenchymal stem cells were detected by flow cytometry.2.108 male SD rats were randomly divided into sham group,CCI-ION group,PBS group,SHED group,SHED+Ig G4 group,and SHED+nivolumab group.The trigeminal nerves of rats in sham group were exposed without ligation,while the chronic compression injury model of trigeminal neuralgia in the CCI-ION group was established.Rats in the PBS group received local injection of PBS on the third day after nerve ligation while those in the SHED group received local injection of SHEDs.Rats in the SHED+Ig G4 group received local injection of Ig G4(homologous control antibody of nivolumab)on day 1,3,5 and 7 after SHED transplantation,and rats in the SHED+nivolumab group received local injection of PD-1 blocking antibody nivolumab on day 1,3,5 and 7 after SHED transplantation.3.The mechanical pain thresholds of rats in the sham group and the CCI-ION group were measured by Von Frey fibers before and 1,3,5 and 7 days after surgery.The mechanical pain thresholds of rats in the PBS group and the SHED group were measured before ligation,before SHED transplantation and 1,3,5,7 days after SHED transplantation.The mechanical pain thresholds of rats in the Ig G4 group and the nivolumab group were measured before antibody injection and at 1,3,5,7 h after antibody injection on 1,3,5 days after SHED transplantation.4.q RT-PCR was used to detect the transcriptional levels of TRPA1 and CGRP m RNA in the TG of rats after nerve ligation in each group.5.Western blot was used to detect the relative protein expression of TRPA1 and CGRP in the TG of rats in each group.6.Double immunofluorescence staining was used to verify the colocalization relationship between CGRP and TRPA1 in the TG of rats.7.Sh-sy5 y cells were cultured and divided into control group,LPS-pretreated group,LPS + SHED coculture group,LPS + SHED co-culture + nivolumab group,LPS + SHED co-culture +Ig G4 group.8.The protein expressional levels of TRPA1 and CGRP in sh-sy5 y cells of each group were detected by western blot.Results: 1.SHED extraction was successful,and the cells were arranged in a swirling pattern.SHEDs were characterized as mesenchymal stem cells by flow cytometry.2.The rat model of trigeminal neuralgia was successfully established.Compared with the sham group,the CCI-ION group had a significant reduction in the mechanical pain threshold on the operated side on the third,fifth,seventh and tenth day(P < 0.0001).3.Compared with the PBS group,the mechanical pain threshold of the SHED group was significantly increased on the third,fifth,seventh day after injection(P < 0.0001、P = 0.0001、P =0.0001).4.Compared with the SHED + nivolumab group,the mechanical pain threshold at 1 and 3 hours after injection of nivolumab in the SHED + Ig G4 group was significantly lower than that in the SHED + Ig G4 group on the first,third and fifth day after SHED transplantation(the first day: P = 0.0028 and P = 0.0003、the third day: P = 0.0007 and P= 0.0029、the fifth day: P = 0.0023 and P = 0.0112).5.Tissue double immunofluorescence staining showed that CGRP and TRPA1 co-expressed in rat trigeminal ganglion neurons.6.The m RNA transcriptional level(P = 0.0021,P = 0.0095)and protein expression level(P = 0.0003、P = 0.005)of TRPA1 and CGRP in trigeminal ganglion of CCI-ION rats increased.7.SHED down-regulated TRPA1 and CGRP m RNA transcription(P = 0.0093,P = 0.0125,respectively)and protein expression(P = 0.0009、P = 0.0444,respectively)in CCI-ION rats.8.Nivolumab reversed the down-regulation effect of SHED on TRPA1 and CGRP m RNA transcription level(P = 0.0004、P < 0.0001)and protein expression(P= 0.001、P = 0.0454).9.The protein expressions of TRPA1 and CGRP in sh-sy5 y cells were increased by LPS pretreatment(P = 0.0058、P = 0.0005,respectively),while SHED co-cultured with sh-sy5 y ce4lls decreased the protein expressions of TRPA1 and CGRP(P= P = 0.0232、P = 0.03,respectively).10,nivolumab attenuated the downregulation of TRPA1 and CGRP induced by SHED co-culture(P = 0.0118、P = 0.001,respectively).Conclusion: Hyperalgesia in rats is associated with the increased m RNA transcription and protein expression of TRPA1 and CGRP in the trigeminal ganglion.SHED alleviates hyperalgesia of trigeminal nerves in rats by downregulating the m RNA transcription and protein expression of TRPA1 and CGRP.Nivolumab,a PD-1 blocking antibody,has a reverse effect on SHEDs’ ameliorative effect on hyperalgesia within 3-5 hours by reproducing the high transcriptional and expressional levels of TRPA1 and CGRP.LPS pretreatment increased the expressional level of TRPA1 and CGRP protein in sh-sy5 y cells.SHED co-cultured with the sh-sy5 y cells can reduce the expression levels of the two proteins mentioned above.Adding nivolumab in the co-culture system of SHED and LPSpretreated sh-sy5 y cells diminished the down-regulation effect of SHED on TRPA1 and CGRP,suggesting that SHED may downregulate the protein expression of TRPA1 and CGRP through paracrine action on PD-1 expressed in neurons of TG and thereby exerting analgesic effect.