The Role Of Follistatin-like Protein 1 In Intervertebral Disc Degeneration And Central Nervous System Inflammation

Part 1:Follistatin-like protein 1 promotes inflammatory reactions in nucleus pulposus cells by interacting with the MAPK and NF?B signaling pathwaysBackground:With the development of society and the prolongation of life expectancy,the incidence of lumbar disease caused by degeneration of intervertebral discs has shown a significant increasing trend.Early observations suggest that the main cause of degeneration is the aging of the intervertebral disc.It can be found that the degeneration of the intervertebral disc has emerged during puberty as the study progresses.These evidences suggest that disc degeneration is not a simple age-related disease and requires more work to explore the mechanism of intervertebral disc disease.Degeneration of the intervertebral disc is accompanied by the decomposition of the extracellular matrix(ECM),which seriously affects the load-bearing capacity of the normal spine and induces low back pain and disc herniation.The occurrence of disc herniation has broken the normal lumbar spine biomechanical relationship,which seriously reduces the patient's quality of life.Although the patient can undergo discectomy and lumbar fusion,the operation also changes the normal structure of the lumbar spine.The cause of accelerated degeneration of the intervertebral disc near the segment,the current treatment of degeneration of the disc has become a hot spot in spinal surgery.The causes of lumbar disc herniation include(1)disc degeneration;(2)lumbar trauma;(3)abnormal spinal structure;(4)genetic factors.The current treatment is not aimed at the pathogenesis of intervertebral disc degeneration,but focuses on relieving the pain caused by degeneration of the intervertebral disc,apparently unable to completely relieve the patient's pain.When degeneration occurs,the local homeostasis of the intervertebral disc tissue is destroyed,and the number of local inflammatory cells and the synthesis of related inflammatory factors change.With the occurrence of degeneration,changes in cells inside the disc include:(1)alternation of cell types in the nucleus pulposus;(2)phenotypic changes in cells;(3)increased density of cells;(4)The cellular activity of the degenerated cells is reduced,which leads to an increase in cell death with abnormal proliferation.These changes ultimately impair the ability of the intervertebral disc cells to synthesize collagen,which ultimately affects the entire intervertebral disc tissue,causing destruction or disintegration of the peripheral nucleus of the nucleus pulposus.These changes lead to the disappearance of the immunological exemption effect of the central nucleus pulposus,the contact of the nucleus pulposus tissue with the peripheral tissue fluid,the stimulation of the body s immune inflammatory response,and the promotion of nucleus pulposus cells and chemotactic inflammatory cells to produce a large number of inflammation-related factors.The combination of local nerve compression and accumulated inflammatory factors causes severe low back pain while inflammatory factors alone can also cause unstressed sciatica.Follistatin-like protein 1(FSTL1),also known as transforming growth factor-beta-stimulated clone 36(TSC-36)or follistatin-related protein(FRP),originally cloned from mouse osteoblasts(MC3T3-E1).FSTL1 is a soluble glycoprotein that is widely expressed in tissues and organs derived from mesenchymal tissues.FSTL1 is involved in the regulation of cell proliferation,apoptosis,metabolism,differentiation,immune response and endocrine function.Studies have shown that in HEK293 cell lines and chondrocytes,FSTL1 mediates inflammation through activated NF?B signaling;in adipocytes and macrophages,FSTL1 increases inflammatory factor expression by activating NF?B and JNK signaling.At the same time,MAPK and NF?B signaling pathways also play a decisive role in local inflammation and degeneration of lumbar intervertebral discs.Studies have confirmed that FSTL1 is closely related to the progression of osteoarthritis,rheumatoid arthritis and various autoimmune diseases,but it is unclear whether FSTL1 plays a role in regulating local inflammation and metabolism of extracellular matrix in intervertebral disc degeneration.This experiment mainly explored the expression of FSTL1 in human and rat intervertebral discs and used the in vitro nucleus pulposus culture model to verify the effect of FSTL1 on nucleus pulposus inflammation and related signaling pathways.Objective:To investigate the role of FSTL1 in intervertebral disc degeneration,we first examined the expression of FSTL1 in patients with intervertebral disc degeneration and degenerative rat intervertebral discs;then human-derived primary nucleus pulposus models were used to explore the mechanism of FSTL1 in the inflammatory response caused by nuclear degeneration in vitro.Methods:(1)Detection of FSTL1 expression in patients with intervertebral disc degeneration and degenerative rat intervertebral discA total of 50 patients with disc herniation who underwent surgery were enrolled and divided into Group P(n = 25)and Group E(n = 25),while recruiting the scoliosis patients(n = 7)as a control.The expression of FSTL1 and related inflammatory factors(TNF-?,IL-1?,MMP-13)in the nucleus pulposus of intervertebral discs in each group were detected.The sera of the patients in Group P,Group E and the normal group(n=14)as control were collected to detect the expression of FSTL1.A rat model of intervertebral disc degeneration was made to detect the expression of FSTL,1 and related inflammatory factors(TNF-?,IL-1?,MMP-13)in degenerated intervertebral disc tissue.(2)Using primary nucleus pulposus models to detect the role of FSTL1 in intervertebral disc inflammation and related mechanismsTo examine the role of FSTL1 in the inflammation of nucleus pulposus cells,the primary nucleus pulposus cell inflammatory model was induced by recombinant human-derived TNF-a,and the mRNA expression and supernatant content of FSTL1 in nucleus pulposus cells were detected.The nucleus pulposus cells were treated with recombinant human-derived FSTL1 to detect changes in mRNA expression and supernatant levels of TNF-a in nucleus pulposus cells.The nucleus pulposus cells were treated with recombinant human-derived FSTL1 to detect the expression of IL-1?,IL-6,COX-2,and iNOS.The nucleus pulposus cells were treated with recombinant human-derived FSTL1,and the expression changes of MAPK and NF?B signaling pathway-related proteins were detected at different time points.Using ERK1/2/MAPK,JNK/MAPK and NF?B signaling pathway inhibitors blocked MAPK and NF?B signaling pathways and detected changes in TNF-?,IL-1?,IL-6,COX-2,iNOS and MMP-13.Results:The expression of TNF-?,IL-1?,and MMP-13 was increased in the nucleus pulposus of the patients with intervertebral disc degeneration and the degenerated nucleus pulposus of the rat.The expression of FSTL1 was increased in the serum and nucleus pulposus of patients in Group P and was positively correlated with the patient's VAS score;the expression of FSTL1 was increased in the degenerated rat intervertebral disc.In TNF-a-induced degenerated intervertebral disc models,FSTL1 protein and mRNA expression increased;expression of inflammatory factors(TNF-a,IL-1?,IL-6,COX-2,iNOS)and matrix degrading enzyme(MMP-13)increased in FSTL1-induced nucleus pulposus cells.In FSTL1-induced nucleus pulposus inflammation model,JNK/MAPK,ERK1/2/MAPK,and NF-kB signaling pathways were activated;and inhibition of JNK/NAPK,ERK1/2/MAPK,and NF?B signaling pathways may reduce FSTLl1-induced expression of inflammatory cytokines.Conclusion:The content of FSTL1 in peripheral blood of patients with degeneration of intervertebral disc was significantly increased,and the content of FSTL1 and VAS score showed a significant positive correlation.In degenerative nucleus pulposus tissue,the expression of FSTL1 was significantly aggravated with degeneration.These results suggest that FSTL1 may be a new marker for the diagnosis of disc disease.FSTL1 promotes intervertebral disc inflammation and nucleus pulposus catabolism by activating JNK/MAPK,ERK1/2/MAPK and NF?B signaling,suggesting that FSTL1 may be a molecular target for the treatment of intervertebral disc degeneration.Part 2:Follistatin-like protein 1 suppressed pro-inflammatory cytokines expression during neuroinflammation induced by lipopolysaccharideBackground:Studies have shown that neuroinflammation is involved in many aspects of the pathological process of neurodegenerative disease.The most important manifestations of neuroinflammatory reactions are the activation of neuroimmune cells,mainly microglia and astrocytes,and the invasion of peripheral immune cells.As the most abundant glial cells in the brain,astrocytes can release a variety of inflammatory mediators,not only cytokines that promote inflammation but also various inflammatory cytokines that inhibit inflammation.Astrocytes can maintain the balance of expression and secretion of proinflammatory and anti-inflammatory cytokines through a variety of regulatory measures,which regulated the immune response of the central nervous system(CNS).Therefore,an inflammation-inducing model of astrocytes is commonly used for the study of central nervous system inflammation.Lipopolysaccharide(LPS),also known as endotoxin,is derived from the outer membrane of Gram-negative bacteria.In vivo application can strongly activate the body's immune system and is commonly used for the induction of inflammation model.LPS promotes chronic inflammatory responses in the central nervous system,causing an increase in beta-amyloid accumulation and functional decline in episodic memory in animal models.These pathological and functional changes can be used to mimic the early stage of the onset of human Alzheimer's Disease(AD).Studies have reported that LPS mediates central nervous system inflammation by up-regulating the expression of various inflammatory cytokines.The representative inflammatory factors are tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6).And IL-1?.Follistatin-like protein 1(FSTL1)is a soluble glycoprotein originally cloned from mouse osteoblast MC3T3-E1 cells,also known as transforming growth factor-?-stimulated clone 36(TSC-36)or follistatin-related protein(FRP).FSTL1 is involved in the regulation of cell survival,proliferation,apoptosis,metabolism,differentiation,immune response and endocrine function.Studies on FSTL1 in inflammatory diseases have continued for more than 20 years,but the specific role of FSTL1 in inflammatory responses remains undecided.Previous studies have shown that FSTL1 plays an important role in the development of the central nervous system in mice from E9.5 to adulthood,but the role of FSTL1 in central nervous system inflammation remains unclear.This study was to investigate the expression of FSTL1 in mice during central inflammation and to investigate the specific role of FSTL1 in inflammatory processes and related mechanisms in vitro.Objective:In this study,we aimed to investigate the effects of FSTL1 on neuroinflammation.We identified changes in the expression of FSTL1 in LPS-induced mouse brain and astrocyte inflammation models.We also explored the specific role of FSTL1 in neuroinflammatory responses and signaling pathways that mediate the expression of related inflammatory factors.Methods:In vivo experiment(1)C57/BJ6 mice was used to prepare inflammation model,and central injection of the lateral ventricle was used to induce a central nervous system inflammatory response by microinjection of 1?L of LPS(5?g/?L).Using the same amount of saline injection group as a control,the expression level of FSTL1 at three-time points of 12h,24h,and 72h was measured,and normal ovarian tissue of mice was used as a positive control for immunohistochemistry of FSTL1.(2)Immunohistochemical staining for local expression of TNF-a,HIF,COX2,IL-1?and IL-6 at three-time points after 12 hours,24h and 72h,and semi-quantitative analysis of protein expression.The tissues used in the TNF-a,HIF,COX2,IL-1?,and IL-6 positive control groups were normal pancreas,kidney,liver,kidney,and pancreas tissues of mice,respectively.In vitro experiments(1)Astrocytes from newborn C57/BJ6 mice were extracted,and the expression of FSTL1 mRNA was detected by LPS treatment at different time gradients(Oh,3h,6h,12h,24h,48h).The expression of FSTL1 protein was measured under concentration gradient(0 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL,1000 ng/mL,1000ng/mL)after LPS treatment.(2)Mouse astrocytes were treated with different concentrations(50 ng/mL,100 ng/mL)of recombinant human-derived FSTL1(hrFSTLl)for 2 h,and then LPS(1000 ng/mL)was added.The effects of different concentrations of FSTL1 on LPS-induced mRNA expression of TNF-?,IL-1?,IL-6 and COX2 were examined.TNF-a expression was quantitatively analyzed using ELISA.Immunofluorescence staining was used to detect the effect of recombinant human-derived FSTL1(hrFSTL1)on LPS-induced TNF-a expression.(3)Mouse astrocytes were treated with recombinant human-derived FSTL1(hrFSTL1;100 ng/mL),Expression of mRNA of TNF-? and IL-1? and IL-6at different time points(Oh,3h,6h,12h,24h,48h)were detected by PCR.Mouse astrocytes were treated with recombinant human-derived FSTL1(hrFSTL1;100 ng/mL),and western blot was used to detect the expression of related proteins in MAPK signaling pathway at different time points(0 min,5 min,15 min,30 min,60 min,120 min).Results:(1)LPS significantly up-regulated the mRNA and protein expression of FSTL1 in mouse brain in a mouse model of neuroinflammation in vitro and in vitro.(2)In the mouse central nervous system inflammation model,immunohistochemcal staining and quantitative analysis showed that the local expression of TNF-a,HIF,COX2,IL-1(3 and IL-6 increased in mouse brain after LPS injection.(3)Pre-addition of hrFSTL1 in LPS-stimulated mouse astrocytes model,the mRNA expression levels of inflammatory factors(TNF-a,IL-1?,IL-6,COX2)were decreased.ELISA showed that pretreatment with FSTL1 significantly inhibited LPS-induced TNF-? expression.(4)Pre-added hrFSTL1 in LPS-stimulated mouse astrocytes,immunofluorescence staining showed that the expression of TNF-a was significantly decreased with the increase of FSTL1 concentration.(5)Adding hrFSTL1 to the astrocyte cell culture model showed that FSTL1 could decrease the mRNA expression of TNF-a,IL-1? and IL-6 in the early stage,but the effect of FSTL1 reversed with the increase of time.Western blot analysis showed that FSTL1 significantly inhibited the activation of the ERK1/2/MAPK signaling pathway.Conclusion:Studies have shown that FSTL1 expression is significantly increased in LPS-induced central inflammatory models in mice and in vitro astrocyte inflammatory models.In an in vitro model,pre-application of FSTL1 inhibits LPS-mediated inflammatory responses in astrocytes.Inhibition of inflammatory response by FSTL1 may be achieved by blocking the ERK1/2/MAPK signaling pathway.This study confirmed that FSTL1 plays a role in inhibiting the expression of inflammatory factors in early neuroinflammation,which provides a new therapeutic approach for the treatment of neuroinflammatory related diseases.