| PART 1 TREM2 Receptor Mediates Microglial Proliferation,Activation and InflammationObjective To investigate the effect of TREM2 on proliferation and activation of microglia and inflammation induced by LPS.Methods The primary culture of neonatal SD rat cerebral cortical microglia was performed.The cells were randomly divided into the following six groups(n=3): control group,LPS-treated(LPS)group,negative-stimulator(scramble+LPS)group,TREM2-small interfering RNA(si RNA+LPS)group,and negative vector(Vector+LPS)group and TREM2 overexpression(pc DNA+LPS)group.Cells in group LPS,scramble+LPS,si RNA+LPS,Vector+LPS and pc DNA+LPS were incubated with LPS(100 ng/m L)for 24 h.Group control did not receive any treatment.In group scramble+LPS group and si RNA+LPS,negative control si RNA and TREM2 si RNA were given 48 h before LPS treatment.Vector virus and TREM2 pc DNA were obtained in Vector+LPS and pc DNA+LPS groups 48 h before LPS treatment.After 24 h of LPS treatment in the six groups of cells,the m RNA levels of TREM2,Iba-1,IL-6,TNF-?,IL-1?,i NOS,IL-4,and IL-10 were determined by RT-PCR.The cell proliferation rate was determined by the method of CCK-8.The apoptosis rate was measured by flow cytometry.The expression level of cleaved caspase 3 was determined by Western blot.Results 1.Compared with the group Control,TREM2 m RNA levels in group LPS,scramble+LPS,si RNA+LPS,Vector+LPS and pc DNA+LPS were significantly increased(P<0.05).Compared with group LPS,TREM2 m RNA level in group si RNA+LPS was significantly lower(P<0.05);TREM2 m RNA level was significantly increased in group pc DNA+LPS(P<0.05).2.Compared with the control group,the proliferation rate and Iba-1 m RNA level of group LPS,scramble+LPS,si RNA+LPS,Vector+LPS and pc DNA+LPS were significantly increased(P<0.05).Compared with the group LPS,the proliferation rate of the group si RNA+LPS and the Iba-1 m RNA level were significantly decreased(P<0.05);the proliferation rate of group pc DNA+LPS and the Iba-1 m RNA level were significantly increased(P<0.05).3.Compared with the control group,the apoptosis rate and cleaved caspase 3 protein levels in LPS group,scramble+LPS group,si RNA+LPS group,Vector+LPS group,and pc DNA+LPS group were significantly increased(P<0.05).Compared with LPS group,apoptosis rate and cleaved caspase 3 protein levels in si RNA+LPS group were significantly higher(P<0.05);apoptotic rate and cleaved caspase 3 protein level in microglia were significantly decreased in pc DNA+LPS group(P< 0.05).4.Compared with the Control group,IL-6,TNF-?,IL-1?,and i NOS m RNA levels in the LPS group,scramble+LPS group,si RNA+LPS group,Vector+LPS group,and pc DNA+LPS group cells were significantly increased(P<0.05),anti-inflammatory cytokines IL-4 and IL-10 m RNA levels were significantly reduced(P<0.05).Compared with LPS group,the levels of pro-inflammatory cytokines TNF-?,IL-1? and i NOS m RNA were significantly decreased in si RNA+LPS group(P<0.05),and the levels of IL-4 and IL-10 m RNA were significantly increased(P< 0.05);pc DNA + LPS group microglial cells pro-inflammatory cytokines TNF-?,IL-1? and i NOS m RNA levels were significantly increased(P <0.05),anti-inflammatory cytokines IL-4 and IL-10 m RNA levels were significantly reduced(P < 0.05).Conclusion The expression of TREM2 increased,the proliferation rate of cells and the expression of activation marker Iba-1 increased,the expression of proinflammatory cytokines increased,and the level of anti-inflammatory cytokines decreased in primary microglia after LPS treatment.TREM2 silencing inhibits proliferation and activation of LPS-treated microglia resulting in inhibition of inflammatory responses.TREM2 overexpression significantly increased the proliferation and activation of LPS-treated microglial cells and further promoted the inflammatory response.PART 2 TREM2 Receptor Elicit Neuropathic Pain via the proliferation and activation of microgliaObjective To investigate the mechanism of TREM2 regulating the proliferation and activation of spinal microglia,mediating inflammatory responses,and participating in neuropathic pain.Methods Sixty SD rats aged 2-3 months were randomly divided into two groups(n=30): sham group and neuropathic pain(NP)group.The CCI model was established in the NP group.The Sham group only exposed the sciatic nerve without ligation.The expression of TREM2 in L4-6 spinal cord of rats in both two groups was detected by Western blot and RT-PCR at 1 d before and 1 d,3 d,7 d,and 14 d after modeling.A number of 72 Sphinx-incorporated successful SD rats were randomly divided into six groups according to the random number table(n=12): Sham group,NP group,TREM2 inhibitor control(NP+Ig G)group and TREM2 inhibitor(NP+antagonist)group,TREM2 agonist control(NP+saline)group and TREM2 agonist(NP+agonist)group.Models of CCI were performed in NP group,NP+Ig G group,NP+antagonist group,NP+saline group and NP+ agonist group.The Sham group only exposed the sciatic nerve without ligation.In rats of NP+antagonist group and NP+Ig G group,TREM2 antibody 0.3ug and Ig G 0.3ug(diluted to 10 ul in PBS)were intrathecally injected respectively 1 d before CCI.In the NP+agonist group and the NP+saline group,Hsp60 5ug and saline 5ug were injected intrathecally at the same time points,and NP group and Sham group were intrathecally injected with an equal volume of saline.All groups were continuously administered for 8 days.The mechanical and thermal pain thresholds of six groups were determined 1 day before modeling and 1d,3d,7d,and 14 d after modeling.After 14-day behavioral testing of CCI,the expression of Iba-1 in the spinal cord of six groups was determined by Western blot.The m RNA levels of inflammatory cytokines IL-6,TNF-?,IL-1?,i NOS,IL-4 and IL-10 were determined by RT-PCR.Immunofluorescence was used to determine the number of microglia in the spinal cord.Results 1.Compared with 1d before CCI model establishment,TREM2 m RNA and protein levels in spinal cord of NP rats increased significantly at 3d,7d,and 14 d after modeling(P<0.05),and the expression was highest at the 3rd day after modeling;TREM2 m RNA and protein of Sham group were highest.There was no significant change at each time point after horizontal modeling(P>0.05).Compared with Sham group,TREM2 m RNA and protein levels in spinal cord of NP rats increased significantly at 3 d,7 d and 14 d after CCI was modeled(P<0.05).2.Compared with Sham group,MPT and TWL in NP group,NP+antagonist group,NP+Ig G group,and NP+saline group were significantly lower on the 3rd,7th,and 14 th day after modeling(P<0.05);MWT and TWL of NP+agonist group had decreased significantly at 1d,3d,7d,and 14 d after modeling(P<0.05).Compared with NP group,MWT and TWL in NP+antagonist group were significantly increased at 3d,7d,and 14 d after modeling(P<0.05);MPT and TWL in NP+agonist group decreased significantly at 1d,3d,7d,and 14 d after modeling(P<0.05).3.Compared with Sham group,the number of spinal microglia and Iba-1 protein in NP group,NP+antagonist group,NP+Ig G group,NP+saline group,and NP+agonist group increased significantly(P<0.05).Compared with NP group,the number of microglia cells and Iba-1 protein in spinal cord of NP+agonist group decreased significantly(P<0.05).The number of microglia Iba-1 protein in spinal cord of NP+agonist group increased significantly(P<0.05).4.Compared with Sham group,the m RNA levels of Tmem119 and P2Ry12 in spinal cord microglia decreased significantly in NP group,NP+antagonist group,NP+Ig G group,NP+saline group,and NP+agonist group(P<0.05).Cst 7 and Spp1 m RNA levels increased significantly(P<0.05).Compared with NP group,the m RNA levels of Tmem119 and P2Ry12 in spinal cord of NP+antagonist rats increased significantly(P<0.05),Cst 7 and Spp1 m RNA levels decreased significantly(P<0.05);Tmem119 and P2Ry12 in spinal cord of NP+agonist rats The m RNA level decreased significantly(P<0.05),and Cst 7 and Spp1 m RNA levels increased(P<0.05).5.Compared with sham group,the IL-6,TNF-?,IL-1?,and i NOS m RNA levels in the spinal cord of rats in NP group,NP+antagonist group,NP+Ig G group,NP+saline group,and NP+agonist group significantly increased(P<0.05);the levels of antiinflammatory cytokines IL-4 and IL-10 m RNA decreased(P<0.05).Compared with NP group,the levels of TNF-?,IL-1? and i NOS m RNA in NP+antagonist rats decreased significantly(P<0.05),and the levels of IL-4 and IL-10 m RNA significantly increased(P<0.05);proinflammatory cytokines TNF-?,IL-1? and i NOS m RNA levels increased in NP+agonist rats(P<0.05),anti-inflammatory cytokines IL-4 and IL-10 m RNA levels decreased(P<0.05).Conclusion TREM2 mediates neuroinflammation,and promotes neuropathic pain via regulating the proliferation and activation of microglia. |
PDF Full Text Request