Study On The Effect Of Downregulating P2X7 Receptor In SGCS Under HGHF Environment On The Expression Of TRPV1 In Neurons

Research background:Purinergic 2X7 receptors（P2X7R）are ATP-gated ion channels that enable the transduction of extracellular signals and causing the corresponding biological effects.P2X7R is involved in cytokine release and regulation of a number of inflammation-related signaling pathways,and its role in inflammation and pain has been extensively studied.Excitation of P2X7R leads to glial cell activation and increased release of glial cell inflammatory factors leading to chronic pain,while inhibition of P2X7R reduces inflammatory pain and chronic neuropathic pain.Transient receptor potential vanilloid receptor 1（TRPV1）is a non-selective cation channel that maintains normal physiological functions of the body.Studies have shown that TRPV1 may play a role in neuronal apoptosis and necrosis.Increased TRPV1activity and expression may promote damage to sensory neurons involved in chronic pain,leading to nociceptive hyperalgesia.Numerous studies have shown that both P2X7R and TRPV1 are closely related to the regulation of pain.In overall animal level experiments,we found that downregulation of P2X7R alleviated nociceptive hyperalgesia in diabetic rats and modulated the release of inflammatory factors in vivo and reduced TRPV1 expression in the dorsal root ganglion（DRG）of diabetic neuropathic pain rats.Therefore,at the cellular level,this project will further explore the effect of downregulation of satellite glial cells P2X7R on neuronal TRPV1 in the environment of high glucose and high free-fat（HGHF）and its possible mechanism,which providing experimental basis and theoretical basis for the pathogenesis of diabetic neuropathic pain.Purpose:In this experiment,we used P2X7 sh RNA to downregulate P2X7R in satellite glial cells（SGCs）in HGHF environment and explored the effect on their neuronal TRPV1expression by coculture.Methods:A model of HGHF-induced injury was constructed on primary cultured satellite glial cells,and the changes of SGCs,neuron-related indexes and receptors were detected after P2X7 sh RNA cell transfection.Experimental groups:normal group（Control）,HGHF model group（HGHF）,HGHF model plus P2X7 sh RNA treatment group（HGHF+P2X7 sh RNA）,HGHF model plus NC sh RNA treatment group（HGHF+NC sh RNA）.（1）Using real time quantitative PCR,we detected the changes in SGCs P2X7 m RNA and neuronal TRPV1 m RNA expression levels.（2）Using immunofluorescence technique,the co-expression levels of P2X7R and glial fibrillary acid protein（GFAP）in satellite glial cells and TRPV1 and neuronal core antigen（Neu N）in neurons were detected.（3）Using western blot analysis,P2X7R,phosphorylated-mitogen-activated protein kinase p38（P-p38）,phosphorylated nuclear transcription factor p65（P-p65）,tumor necrosis factorɑ（TNF-α）,interleukin 1-β（IL-1β）in each group of SGCs and TRPV1,protein kinase C-?（PKC-?）and P-p38 in neurons were detected.（4）The relative levels of Ca²⁺in SGCs and neurons were measured by BBcell Probe F03.（5）The levels of TNF-αand IL-1βreleased from SGCs were measured by Enzyme-linked immunosorbent assay（ELISA）.Results:1.（1）Compared with the Control group,the P2X7R transcript level（P<0.01）and protein level（P<0.001）of SGCs in the HGHF group were significantly increased.Compared with the HGHF group,the P2X7R transcript level（P<0.01）and protein level（P<0.001）of SGCs in the HGHF+P2X7 sh RNA group were significantly decreased.（2）SGCs P2X7R and GFAP co-expression,the highest degree of co-expression of GFAP and P2X7R in HGHF group（P<0.001）,and the degree of co-expression in HGHF+P2X7 sh RNA group was lower than that in HGHF group（P<0.001）.2.（1）Compared with the Control group,the neuronal TRPV1 transcript level（P<0.001）and protein level（P<0.01）were significantly increased after co-culture with SGCs in the HGHF group,and the neuronal TRPV1 transcript level（P<0.001）and protein level（P<0.01）were significantly decreased.（2）Neuronal TRPV1 and Neu N co-expression,the highest degree of neuronal TRPV1 and Neu N co-expression after co-culture with SGCs of HGHF group（P<0.01）,and the lower degree of neuronal co-expression after co-culture with SGCs of HGHF+P2X7 sh RNA group than HGHF group（P<0.01）.3.（1）Compared with the Control group,the expression of P-p38 and P-p65 proteins in satellite glial cells in the HGHF group was significantly higher（P<0.001）.Compared with the HGHF group,the expression of P-p38 and P-p65 proteins in the HGHF+P2X7sh RNA group was significantly lower（P<0.01）.（2）Compared with the Control group,neuronal PKC-?and P-p38 proteins were significantly increased after co-culture with SGCs in the HGHF group（P<0.01）.Neuronal PKC-?and P-p38 proteins were significantly decreased after co-culture with SGCs in the HGHF+P2X7 sh RNA group compared with the HGHF group（P<0.01）.4.（1）Ca²⁺content of SGCs in HGHF group was higher than that in Control group（P<0.001）,while Ca²⁺content of SGCs in HGHF+P2X7 sh RNA group was lower than that in HGHF group（P<0.01）.（2）Ca²⁺content of neurons after co-culture with SGCs in HGHF group was higher than that in Control group（P<0.01）.The neuronal Ca²⁺content after co-culture with SGCs in HGHF+P2X7 sh RNA group was lower than that in HGHF group（P<0.01）.5.（1）Compared with the Control group,the expression of IL-1β（P<0.001）and TNF-α（P<0.01）proteins in SGCs in the HGHF group was significantly higher.Compared with the HGHF group,the expression of IL-1βand TNF-α（P<0.01）proteins in the HGHF+P2X7 sh RNA group was significantly lower.（2）Compared with the Control group,the release of inflammatory factors IL-1βand TNF-αby SGCs in the HGHF group was significantly increased（P<0.01）.Compared with the HGHF group,the release of inflammatory factors IL-1βand TNF-αby SGCs in the HGHF+P2X7 sh RNA group was significantly decreased（P<0.01）.6.The expression of GFAP was elevated in SGCs induced by HGHF environment（P<0.001）,while downregulation of P2X7R with P2X7 sh RNA reduced the rise of GFAP expression induced by HGHF（P<0.01）.Conclusion:HGHF induces activation of SGCs and increases inflammatory factor release,downregulates P2X7R in SGCs in HGHF environment and reduces neuronal TRPV1expression;Ca²⁺/P38 MAPK/NF-κB signaling pathway may be involved in the process of downregulating P2X7R to reduce inflammatory factor release in SGCs,while Ca²⁺/PKC-?/P38 MAPK signaling pathway may be involved in the pathophysiological process of downregulating P2X7R to inhibit neural TRPV1 expression.