The Role And Mechanism Of TREM2 Mediating Macrophage Autophagy In The Regulation Of Neuropathic Pain Through MTOR Signaling Pathway

Objective: Neuropathic pain(NP)is a common clinical disease,the specific pathogenesis has not been clarified,there is a lack of specific treatment,which seriously affects the quality of life of patients.Therefore,it is urgent to find new treatment strategy for NP.Macrophages are widely involved in the regulation of various life activities of the body,and play a key role in the pathological process of NP.Triggering receptor expressed on myeloid cells-2(TREM2)is mainly expressed in peripheral macrophages and microglia in the central nervous system.Numerous studies have shown that TREM2 is involved in the regulation of NP by mediating microglial autophagy.Macrophage autophagy is associated with pathological processes of various diseases.However,it has not been shown whether TREM2 can regulate the development of NP by mediating autophagy in peripheral macrophages.In order to clarify the regulatory role of TREM2 in the pathology of NP,we constructed NP model-spared nerve injury(SNI)model and detected the expression of TREM2 in sciatic nerve tissue of mice before and after modeling.Then,by comparing the mechanical pain threshold of TREM2 gene knockout mice(Trem2-/-mice)and wild type mice(WT)after modeling,the regulatory effect of TREM2 on NP was clarified.Finally,the expression levels of P62,LC3,m TOR,p-m TOR,ULK1 and p-ULK1 in sciatic nerve tissue of Trem2-/-mice and WT mice after modeling were compared to explore the related mechanism of TREM2’s regulation of NP.To explore the mechanism of NP,we used the sciatic nerve grinding fluid from WT mice to stimulate RAW264.7 cells to establish a cell model of NP and detected the expression of TREM2 in macrophages.The RAW264.7 cells were transfected with TREM2 si RNA adenovirus to silence the TREM2 gene.The autophagy level of RAW264.7 cells was detected to further clarify the effect of TREM2 on macrophages and its related mechanism.Methods: In the animal experiments,firstly,male,8w,C57BL/6 mice were induced anesthesia,and SNI model or Sham operation(Sham)model were established.The mechanical pain threshold baseline(BL)of mice in each group was measured before surgery and at the 7th,14 th and 21 th day after surgery.After behavioral tests,sciatic nerve tissues of mice in both surgical and non-surgical sides were taken from each group,and the expression of TREM2 was determined by RT-PCR and western blotting.Then,male,8w,Trem2-/-mice and WT mice were used to establish SNI models.The ipsilateral and contralateral mechanical pain thresholds of SNI mice were measured before modeling(BL)and on days 7,14 and 21 after modeling.Then,the ipsilateral and contralateral sciatic nerves of Trem2-/-mice and WT mice were collected on the 7th day after SNI,and the expression levels of P62 and LC3 proteins were detected by immunoblotting,and the cellular distribution of P62 protein expression was determined by immunofluorescence technique.Finally,we took the ipsilateral and the contralateral sciatic nerve injury segment of SNI in Trem2-/-mice and WT mice on day 7 of SNI,and determined the protein expression levels of m TOR,p-m TOR,ULK1,and p-ULK1 in the sciatic nerve tissue by immunoblotting.In the cytological experiments,the expression of TREM2 m RNA was measured by RT-PCR after the RAW264.7 macrophage cell line,which was in good condition and about 60% confluence,was stimulated with the sciatic nerve grinding solution of WT mice or PBS for 36 hours.The RAW264.7 TREM2 gene was silenced by adenovirus vector carrying TREM2 si RNA,and the cell model was constructed according to the above methods.The macrophage cell line was taken 36 hours after modeling,and the expression levels of P62 and LC3 protein in RAW264.7 were detected by Western blot.Results: The neuropathic pain model of mice was successfully constructed by ligation and cutting the tibial nerve and the common peroneal nerve,which were two branches of the sciatic nerve in mice.The mechanical pain threshold of mice was significantly reduced,and the expression level of TREM2 in the sciatic nerve on the ipsilateral side of SNI was significantly increased.Compared with WT group,the ipsilateral mechanical pain threshold of Trem2-/-group was significantly increased.Two groups of the contralateral mechanical pain threshold is no significant difference;At 7 days after modeling,compared with the WT group,the expression of P62 protein in the ipsilateral sciatic nerve tissue of Trem2-/-mice was significantly decreased,and the LC3Ⅱ/LC3Ⅰ ratio was significantly increased.The level of m TOR activation was lower and the level of ULK1 activation was higher.There was no significant difference in the protein expression of contralateral sciatic nerve tissue between the two groups.The expression of TREM2 in RAW264.7 cells was significantly increased after 36 hours of stimulation with sciatic nerve grinding solution.The silencing efficiency of TREM2 gene was up to 80% at 72 h after transfection of TREM2 si RNA adenovirus.After stimulation with sciatic nerve grinding solution for 36 h,the expression of P62 was significantly decreased,and the LC3Ⅱ/LC3Ⅰ ratio was significantly increased.Conclusion: The results suggest that TREM2 is involved in the regulation of neuropathic pain,and the mechanism may be related to the activation of m TOR and the inhibition of ULK1 activation,thus inhibiting macrophage autophagy.