Based On The Study Of Tollip And TLR4 Expression Level And Its Correlation In Spinal Tuberculosis

Objective:Expression levels of Toll Interacting Protein(Tollip)and Toll-like Receptor 4(TLR4)were measured quantitatively and qualitatively in three tissues of spinal tuberculosis(STB)focus tissue,STB parafocus tissue,and non-tuberculous tissue,And explore the correlation between Tollip and TLR4 expression in these same tissue.The possible important role of negative feedback regulator Tollip in STB immune response was preliminarily explained.Methods:A total of 15 STB patients admitted to the Affiliated Hospital of Youjiang Medical University for Nationalities for concurrent surgery from December 2019 to November 2020 were selected,During the operation,STB focus tissue(tuberculosis focus group)and STB parafocus tissue(tuberculosis parafocus group)were collected respectively.In the same period,13 patients with lumbar spinal stenosis(LSS)were treated by surgery.The decompression tissue of the spinal canal was collected intraoperatively(non-tuberculosis group).Western blot(WB)and real-time quantitative polymerase chain reaction(RT-q PCR)were used to detect the protein expression levels and mRNA relative expression levels of Tollip and TLR4 in the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group,respectively.Then the expression differences of Tollip and TLR4among the three groups and the correlation between Tollip and TLR4 in protein level and mRNA level were analyzed.In addition,the expression levels of Tollip and TLR4 were qualitatively detected by immunohistochemistry(IHC)in the tuberculosis focus group,tuberculosis parafocus group and non-tuberculosis group,the expression differences between the groups and the correlation between the two proteins were analyzed.Results:(1)WB test results:The Tollip protein expression levels of the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group were429.19±160.75,319.68±162.46,[5.96(2.96,326.80)].There was significant difference between the three groups(χ~2=13.21,P<0.001).The expression level of Tollip protein in the tuberculosis focus group was significantly higher than that in the non-tuberculosis group(χ~2=10.55,P=0.001).The TLR4 protein expression levels of the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group were 686.25±98.25,[199.08(118.70,460.85)],[14.82(5.35,198.29)].There was significant difference between the three groups(χ~2=24.07,P<0.001).The expression level of TLR4 in the tuberculosis focus group was significantly higher than that in the tuberculosis parafocus group(χ~2=11.43,P=0.001)and the non-tuberculosis group(χ~2=18.55,P<0.001),The tuberculosis parafocus group was significantly higher than the non-tuberculosis group(χ~2=6.54,P=0.011).Correlation analysis of Tollip and TLR4 protein expression levels:There was a negative correlation between Tollip and TLR4 protein expression in the tuberculosis focus group(r=-0.725,P=0.002).There was a positive correlation between Tollip and TLR4 protein expression in non-tuberculosis group(r=0.703,P=0.007).(2)Results of RT-q PCR:The mRNA relative expression levels of Tollip in the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group were[1.85(1.00,4.18)],[1.00(0.43,1.20)],[0.52(0.44,1.26)].There was significant difference between the three groups(χ~2=11.98,P=0.003).The mRNA relative expression levels of Tollip in the tuberculosis focus group was significantly higher than that in the tuberculosis parafocus group(χ~2=6.22,P=0.013)and the non-tuberculosis group(χ~2=10.41,P=0.001).The mRNA relative expression levels of TLR4 in the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group were[2.67(1.04,6.91)],[1.00(0.47,1.02)],[0.36(0.24,0.82)].There was significant difference between the three groups(χ~2=18.34,P<0.001).The mRNA relative expression levels of TLR4 in the tuberculosis focus group was significantly higher than that in the tuberculosis parafocus group(χ~2=10.38,P=0.001)and the non-tuberculosis group(χ~2=12.26,P<0.001),The tuberculosis parafocus group was significantly higher than the non-tuberculosis group(χ~2=5.89,P=0.015).Correlation analysis results of Tollip and TLR4 mRNA relative expression levels:The mRNA relative expression levels of Tollip and TLR4 were positively correlated in the tuberculosis parafocus group(r=0.687,P=0.005).The mRNA relative expression levels of Tollip and TLR4in the non-tuberculosis group were positively correlated(r=0.855,P<0.001).(3)IHC results:There was no significant difference(χ~2=4.489,P=0.344)in the low,moderate and high expression of Tollip among the tuberculosis focus group(6.67%,1/15),(53.33%,8/15),(40.00%,6/15),the tuberculosis parafocus group(33.33%,5/15),(46.67%,7/15),(20.00%,3/15)and the non-tuberculosis group(15.38%,2/13),(61.54%,8/13),(23.08%,3/13);There was no significant difference(χ~2=3.508,P=0.477)in the low,moderate and high expression of TLR4 among the tuberculosis focus group(13.33%,2/15),(66.67%,10/15),(20.00%,3/15),the tuberculosis parafocus group(26.67%,4/15),(66.67%,10/15),(6.66%,1/15)and the non-tuberculosis group(23.08%,3/13),(46.15%,6/13),(30.77%,4/13);In the IHC test results,There was no statistical significance in the correlation analysis of Tollip and TLR4expression levels in the tuberculosis focus group,the tuberculosis parafocus group and the non-tuberculosis group(χ~2=2.833,P=0.586;χ~2=5.486,P=0.241;χ~2=7.438,P=0.114).Conclusion:(1)The protein expression levels and mRNA relative expression levels of Tollip and TLR4 in STB focus tissue were significantly higher than those in STB parafocus tissue and non-tuberculous tissue.It is suggested that Tollip may play an important role in the immune response to STB lesions.(2)In the group with statistically significant correlation,Tollip and TLR4 in STB focus tissue were negatively correlated at protein levels.Tollip and TLR4 were positively correlated at protein and mRNA levels in non-tuberculous tissue,In addition,there was a positive correlation between Tollip and TLR4 protein levels in STB parafocus tissue.It is suggested that Tollip may have a negative feedback regulation effect on TLR4 signal transduction pathway during the occurrence and development of STB,and the effect of Tollip on STB focus tissue is more obvious than that on spinal tuberculosis para-focal tissues and non-tuberculous tissue.