Mechanism Study Of ATF6-Regulate Inflammatory Response In Intervertebral Disc Degeneration

Background: Low back pain is a common symptom of the skeletal muscle system.It is estimated that approximately 540 million people around the world have been affected by low back pain which placing a huge burden on public health and socio-economic development.More than 90%of LBP with unclear etiologies so far,precise and effective treatments are scarce.It is widely believed that intervertebral disc degeneration(IVDD)is the most common change associated with low back pain.The intervertebral disc is a complex joint,which is composed of gel-like tissue in the center(rich in proteoglycan and type II collagen,called nucleus pulposus,NP),collagen-rich fibrous layered structures(called annulus fibrosus AF)surrounding the nucleus pulposus and cartilage endplates(Cartilage endplates CE).Various factors lead to an imbalance between extracellular matrix(ECM)anabolism and catabolism,inducing ECM degradation,which is a characteristic change in intervertebral disc degeneration and generally irreversible.In recent years,many studies have shown that inflammatory response is an important cause of IVDD,inflammatory cytokines such as tumor necrosis factor(TNF-α)and interleukin(IL)are closely related to ECM degradation and intervertebral disc degeneration.Therefore,a deeper exploration of the mechanism of inflammatory response in nucleus pulposus cells may bring new ideas for the treatment of intervertebral disc degeneration.Objective: To explore the specific mechanism of activating transcription factor 6(ATF6)in regulating the inflammatory response of nucleus pulposus tissue and mediating IVDD,further elaborate the key role of inflammatory response in intervertebral disc degeneration,and provide new ideas for its treatment.Method:(1)Downloaded the data of intervertebral disc nucleus pulposus from the GEO database,used bioinformatics analysis methods to find the differentially expressed gene ATF6 related to inflammation in degenerative intervertebral disc tissues.Immunohistochemical staining,RT-PCR,and immunoblotting were used to detect the expression of ATF6 in intervertebral disc tissue and its correlation with IL-1β and TNF-αexpression.(2)Primary nucleus pulposus cells were extracted,identified,and cultured.LPS was used to simulate the pathological changes of inflammation and the extracellular matrix degradation in nucleus pulposus cells during intervertebral disc degeneration.Cellular immunofluorescence,RT-PCR,and immunoblotting were used to detect the inflammatory level of nucleus pulposus cells;Cell transfection technology was used to suppress or overexpress ATF6 in nucleus pulposus cells.Cell immunofluorescence and immunoblotting were used to detect LPSinduced inflammation levels and extracellular matrix changes in nucleus pulposus cells.Using chromatin immunoprecipitation sequencing technology to screen genes that bind to ATF6 in the promoter region.The binding site of the target gene and ATF6 was predicted using the JASPER database;the relationship between the target gene and its downstream pathway and ATF6 was verified by chromatin immunoprecipitation,cell transfection,RT-PCR and western blot.(3)A rat model of lumbar intervertebral disc degeneration was established using annulus fibrosus puncture,and the effect of ATF6 on intervertebral disc degeneration was investigated by local injection of ATF6 small interfering RNA.Magnetic resonance imaging,H&E staining,Masson staining,histopathological scores,and immunohistochemical staining were used to evaluate the degree of intervertebral disc degeneration in rats,and the role of ATF6 in intervertebral disc degeneration in vivo was explored.Result:(1)Immunohistochemical staining,RT-PCR,and immunoblotting results showed that compared with control nucleus pulposus tissue,the degenerated nucleus pulposus tissue of the intervertebral disc contained more ATF6 、 IL-1β and TNF-α.The expression of ATF6 in degenerated nucleus pulposus tissue was relevant with IL-1β and TNF-α.(2)Cellular immunofluorescence showed that primary nucleus pulposus cells were successfully extracted,and LPS could induce the inflammatory environment and extracellular matrix degradation during intervertebral disc degeneration;Inhibiting ATF6 gene in nucleus pulposus cells alleviated LPS-induced inflammation and extracellular matrix degradation in nucleus pulposus cells;conversely,overexpression of ATF6 gene aggravated LPS-induced inflammation and extracellular matrix degradation in nucleus pulposus cells.Chromatin immunoprecipitation sequencing and chromatin immunoprecipitation confirmed that ATF6 can bind to the promoter region of MEFV gene and accelerate the expression of MEFV;ATF6 activated more Caspase-1 by promoting the expression of MEFV,thereby producing more IL-1β,which promoted the inflammation of nucleus pulposus cells and degradation of extracellular matrix.(3)Animal model of lumbar disc degeneration in rats was successfully established using annulus fibrosus puncture.It was demonstrated by magnetic resonance imaging,histopathology,and immunohistochemistry that inhibition of ATF6 can alleviate lumbar disc degeneration induced by annulus fibrosus puncture in rats.Conclusion:ATF6 can bind to the promoter region of MEFV gene and promote its transcription activation,regulate the inflammatory response of nucleus pulposus cells and exacerbate extracellular matrix degradation,mediating intervertebral disc degeneration.