Study On The Activation Of Schwann Cell Inflammatory Signal Pathway By Saturated Fatty Acids In Sciatic Nerve Injury In Rats

Objective Sciatic nerve injuries are commonly encountered diseases caused by traffic accidents,industrial injuries and operations,which seriously threat human health.The peripheral nervous system needs a lot of saturated fatty acids to make it myelinated,so a large amount of free saturated fatty acids will be released after sciatic nerve injury.Saturated fatty acids can trigger microglia and astrocytes to release inflammatory cytokines through TLR4/MD2 signal pathway,but its role in sciatic nerve injury is still unclear.It is of important clinical benefit to explore whether saturated fatty acids activate Schwann cell TLR4/MD2 signal pathway to trigger inflammation in injured sciatic nerve.Methods Cell experiments: 1.The effect of palmitic acid(PA)on the viability of Schwann cells was explored by CCK8 experiment.The mRNA expression levels of inflammatory cytokines Tnfa and Il-6 in control group and PA group were detected by RT-q PCR.The protein secretion levels of TNFα and IL-6 in the control group and PA group were detected by ELISA.And the expression of TLR4/MD2 pathway related proteins was detected by Western Blot.2.The effect of inhibition of TLR4,MD2,NF-κB or MAPKs on the secretion of inflammatory cytokines induced by PA in Schwann cells was detected by ELISA.Protein changes of My D88,p-p65,P65,p-JNK,JNK,p-ERK,ERK,p-p38 and P38 were detected by Western Blot after using TAK242,an inhibitor of TLR4,or L6H21,an inhibitor of MD2.Animal experiments:Immunofluorescence staining was used to detect the frequency of Schwann cells.RT-q PCR was used to detect the expression of inflammatory cytokines Tnfa,Il-1b and Il-6 mRNA.The levels of inflammatory cytokines TNFα,IL-1β and IL-6 were detected by Western Blot.HE staining was used to detect inflammatory cell infiltration.Results Cell experiments: 1.The results of CCK8 showed that compared with the control group,50 and 100 μM PA had little effect on Schwann cells,while 150 and 200μM PA had certain cytotoxicity to Schwann cells,so the concentration of PA in subsequent experiments was 100 μM.RT-q PCR results showed that the mRNA expression of Tnfa and Il-6 in Schwann cells increased after 6 hours of PA stimulation.The results of ELISA showed that TNFα and IL-6 secreted by Schwann cells significantly increased after 24 hours of PA stimulation.The results of Western Blot showed that PA activated TLR4/MD2 signaling pathway,and the expression of TLR4,MD2,My D88,NF-κB and MAPKs protein increased.2.Blocking TLR4 or MD2 can inhibit the secretion of TNFα and IL-6 inflammatory factors in Schwann cells induced by PA.Blocking NF-κB can inhibit TNFα secretion of Schwann cells induced by PA,but has no effect on IL-6 secretion.Blocking MAPKs can inhibit the secretion of TNFα and IL-6inflammatory factors in Schwann cells induced by PA.The results of Western Blot showed that blocking TLR4 or MD2 could inhibit the expression of My D88,p-P65,p-JNK,p-ERK and p-P38 induced by PA.Animal experiments: The results of immunofluorescence staining showed that the frequency of Schwann cells increased 24 hours after sciatic nerve injury,and blocking MD2 or TLR4 inhibited the frequency of Schwann cells.Compared with injured group,the mRNA expression levels of Tnfa,Il-1b and Il-6 in L6H21 group were significantly lower.The mRNA expression levels of Tnfa and Il-6 in TAK242 group were significantly lower than those in injured group,but the mRNA level of Il-1b had no change.The results of Western Blot showed that compared with injured group,the expression of TNFα,IL-1β and IL-6 protein in L6H21 group and TAK242 group decreased significantly 24 hours after sciatic nerve injury,HE staining showed that the sciatic nerve fibers of exposed group were neat and clear,and no inflammatory cells were found,but inflammatory cells were found in injured group,L6H21 group and TAK242 group.The infiltration of inflammatory cells in L6H21 group and TAK242 group was significantly less than that in injured group.Conclusion 1.Saturated fatty acids can activate the inflammatory response of Schwann cells through the TLR4/MD2 pathway,which provides a basis for further exploring the mechanism of inflammatory response caused by sciatic nerve injury.2.The model of sciatic nerve injury in rats was successfully constructed,which laid a foundation for the study of animal experiments and revealed that TLR4 and MD2 are involved in the occurrence of inflammatory response after sciatic nerve injury.