The Mechanism Study Of IL-6/JAK1/STAT3 Pathway Involved In Intervertebral Disc Resorption By Regulating The Expression Of TSLP

Objective: To investigate the mechanism of interleukin-6(IL-6)promoting the process of herniated lumbar disc resorption via upregulating the expression of thymic stromal lymphopoietin(TSLP)through JAK1/STAT3 signaling pathway in NP cells.Methods: Primary rat NP cells were isolated and cultured in vitro.Immunofluorescence staining was used to detect the expression of type II collagen in primary cells.The third generation of well-growing rat NP cells were randomly divided into three groups.The NP cells of each group were treated with 10ng/m L recombinant Rat-IL-6(IL-6 group)or20μM STAT3 inhibitor BP-1-102(BP group)for 3～72 hours.The control group was only cultured with DMEM/F12 medium under the same conditions.The gene expressions of IL-6R,Jak1,STAT3,TSLP,and MMP-3 in NP cells were detected by q RT-PCR.The effects of IL-6 on JAK/STAT signaling pathway factors and resorption-related factors such as TSLP and MMP-3 were analyzed.The protein expression of p-STAT3 and TSLP in each group was detected by Western blot to verify the mechanism of IL-6 upregulating the expression of TSLP through the JAK1/STAT3 signaling pathway and promoting resorption.Results: The primary rat NP cells were triangular and polygonal under a fluorescence microscope,with good refraction.Under a fluorescence microscope,type II collagen was yellow-green fluorescence,mainly concentrated in the cytoplasm.DAPI staining of the nucleus was obvious as blue fluorescence.According to previous reports,the isolated cells were identified as rat NP cells.the recombinant Rat-IL-6(10ng/m L)was used to stimulate rat NP cells.The results of q RT-PCR showed that: IL-6R gene was highly expressed in the first 3 hours(3h-IL-6R: 2.86±0.20,control group-IL-6R:1.02±0.03,P<0.05),Then it began to decline,and at 6-72 hours the gene expression of IL-6R maintained a relatively stable high expression,and the differences among the groups were statistically significant compared with the control group;while JAK1 and STAT3 genes gradually increased after 6 hours stimulation(6h-JAK1: 1.51±0.10,control group-JAK1: 1.02±0.02,P<0.05;6h-STAT3: 2.05±0.10,control group-STAT3:0.96±0.09,P<0.05),reaching the peak at 12h(12h-JAK1: 2.28±0.23,control group-JAK1: 1.02±0.02,P<0.05;12h-STAT3: 5.38±0.76,control group-STAT3:1.09±0.08,P<0.05),then it began to decrease but all of them were higher than the control group,the difference was statistically significant.The expression of TSLP was similar to that of JAK1 and STAT3,and increased gradually after stimulation by IL-6,reaching the peak at 12h(12h-TSLP:2.85±0.08,control group-TSLP:1.01±0.01,P<0.05).Compared with JAK1,STAT3 and TSLP,the peak time of MMP-3 was significantly delayed to 24 hours(24h-MMP-3: 4.54±0.24,control group-MMP-3:1.02±0.07,P<0.05),and then maintained at a high level.The results showed that 12 hours was the strongest time point of IL-6 on rat NP cells.The expression of STAT3 and TSLP in NP cells was detected after adding STAT3 inhibitor bp-1-102 for 12 hours.q RT-PCR results showed that compared with the control group(control group-STAT3:1.08±0.15;control group-TSLP: 0.98±0.08),the expression levels of STAT3 and TSLP in BP group were significantly decreased(IL-6 group-STAT3: 0.19±0.02,BP group-TSLP: 0.08±0.003,P<0.05).Western blot results showed that: after IL-6stimulated NP cells for 12 hours,the protein expression of p-STAT3 and TSLP in the IL-6 group was significantly higher than that in the control group,while the protein expression of p-STAT3 and TSLP in the BP group was significantly lower than that in IL-6 group.Conclusion: 1.IL-6 can upregulate the expression of TSLP and MMP-3 in rat NP cells to promote the process of resorption.2.IL-6 can upregulate the expression of TSLP through JAK1/STAT3 signaling pathway to promote the resorption of herniated intervertebral disc.3.Rat NP cells can quickly respond to IL-6 stimulation,and the best treated time is 12 hours.