Role And Potential Mechanism Of Arg2 In Intervertebral Disc Degeneration

BackgroundLow back pain(LBP)is one of the most primary symptoms of the lower back and may be accompanied by radiation pain or numbness in the lower extremities.LBP is the most common musculoskeletal symptom,affecting the quality of life and resulting in a huge burden to thewhole society.Although many countries invest a lot of medical resources into the prevention and treatment of LBP every year,this problem remains unsolved.Intervertebral disc(IVD)degeneration(IVDD)has been reported to be the first leading cause of LBP.Although a great deal of studies on IVDD have been conducted,the molecular mechanisms of IVDD remain unclear.Patients with IVDD will develop severe spinal degeneration with irreversible neurological impairment if we fail to treat the primary causes of IVDD.Therefore,elucidating the potential mechanisms of IVDD is helpful in figuring out simple and effective treatment and prevention methods.IVDD is closely related to nucleus pulposus(NP)cells(NPCs)dysfunction.NPCs,affected by various stimuli,undergo a series of pathophysiological changes: accelerated senescence,increased apoptosis,impaired mitochondrial function and increased level of reactive oxygen species,which further jeopardize NPCs,reduce the production of ECM and raise the level of ECM degradation related enzymes.Eventually,the decreased number and the impaired function of NPCs can be observed,which lead to the trigger or aggravation of IVDD.Arginase help the transformation of arginine into ornithine and urea.Arginase II(Arg2),one of the primary forms of arginase,is mainly expressed in extrahepatic tissues.Arg2 plays a pivotal tole in regulating cellular aging,cell death,and inflammatory response.Previously,Arg2 has been shown to increase the level of matrix metalloproteinases(MMPS)in chondrocytes,leading to cartilage tissue destruction and osteoarthritis.However,the role and related mechanism of Arg2 in IVDD have not been studied so far.Due to the biological similarities between osteoarticular tissues and intervertebral disc tissues,as well as the similarities of development between osteoarthritis and IVDD,we hypothesized that the disordered expression of Arg2 might be involved in the development of IVDD.Objectives1.To investigate the expression of Arg2 in degenerative nucleus pulposus tissues,and to reveal the correlation between Arg2 and IVDD;2.To investigate therole of Arg2 in apoptosis,senescence,oxidative stress and degeneration of NPCs and the potential pathways;3.To verify the effect of Arg2 on IVDD in animals.Part Ⅰ.Correlation between Arg2 and intervertebral disc degeneration.Methods:(1)Pfirrmann grading was performed according to the MRI findings;(2)NP tissues of IVD were obtained;(3)Cellular immunofluorescence was utilized to detect the degeneration indicators;(4)Immunohistochemistry,Western Blot,RT-q PCR and ELISA were used to assess the expression of Arg2 in NP tissues;(5)The primary NPCs were extracted and identified;(6)IL-1β was used to treat NPCs to establish the degenerative cell model and the model was verified;(7)The expression level of Arg2 in the degenerative cell model was detected by RT-q PCR.Results:(1)The degenerated IVD was graded based on Pfirrmann grading criteria;(2)Immunohistochemistry,Western Blot and RT-q PCR results revealed that Arg2 in the degenerative IVD was dramatically increased when compared with that in normal IVD,and besides,the Arg2 level increased with the severity of IVDD.(3)Arg2 level in NPCs was measured by ELISA,and then the correlation between Arg2 level and Pfirrmann scores was analyzed.The results demonstrated that Arg2 expression level was correlated with Pfirrmann scores of IVDD.(4)Primary NPCs were successfully isolated and were used for the degenerative model.(5)Arg2 expression level was increased in degenerative NPCs.Conclusions: In this part of the study,it was found that Arg2 expression level was increased in degenerative intervertebral discs.In the cell model of degeneration constructed with IL-1β,Arg2 expression level was also significantly increased.Part Ⅱ.Arg2 affects apoptosis,senescence,oxidative stress and degeneration of NPCs by regulating the NF-κB pathway.Methods:(1)The si-RNA of Arg2(siArg2)was established and siArg2 was used to interrupt the expression of Arg2;(2)Western Blot and immunofluorescence helped assess the effects of si Arg2 on ECM,senescence,apoptosis and oxidative stress of NPCs;(3)Arg2 overexpression plasmid was constructed,and the changes of ECM degradation,senescence,apoptosis and oxidative stress of NPCs were detected after Arg2 was overexpressed;(4)After specifically blocking the NF-κB pathway by JSH-23,the expression of Arg2 and the changes in ECM degradation,senescence,apoptosis and oxidative stress of NPCs were detected.Results:(1)siArg2 significantly reduced the expression level of Arg2;(2)siArg2 improved ECM synthesis and lowered oxidative stress,senescence and apoptosis of NPCs;(3)Arg2 overexpression exacerbated IL-1β-induced ECM degradation,oxidative stress,senescence and apoptosis of NPCs;(4)The NF-κB pathway was overactivated after Arg2 level was raised;(5)Blocking the NF-κB pathway using JSH-23 improved the balance of ECM synthesis and catabolism in NPCs,and ameliorated oxidative stress,senescence and apoptosis of NPCs.Conclusions: In this part,si-RNA and plasmid were used to regulate the level of Arg2,and the results revealed that Arg2 helped regulate ECM metabolism and ameliorated oxidative stress,senescence and apoptosis of NPCs.In addition,Arg2 regulated the metabolism of nucleus pulposus ECM and modulated oxidative stress,senescence and apoptosis of NPCs through the NF-κB pathway.Part Ⅲ.si-Arg2 ameliorated IVDD in rats.Methods:(1)Establishing an animal model of IVDD;(2)si-Arg2 or control reagent was injected into discs;(3)The severity and structure of IVDD was assessed using safranin fast green staining;(4)The indicators of IVDD were detected using immunohistochemistry.Results:(1)The animal model of IVDD was successfully constructed;(2)The staining results showed that si Arg2 increased the content of NP tissue;(3)Immunohistochemistry results showed that NPCs with collagen type II in IVDD+si Arg2 group was significantly raised than that in IVDD group and IVDD+si-NC group,and NPCs with MMP3 in IVDD+si Arg2 group was significantly lower than that in IVDD group and IVDD+si-NC group.Conclusions: In this part of the study,si-Arg2 significantly alleviated IVDD and ameliorated ECM degradation in rats.SummaryThis present research explored the role of Arg2 in the development of IVDD.Various molecule biology techniques showed that Arg2 level was increased in degenerated IVD.Regulation of Arg2 by si-RNA and plasmid showed that Arg2 could aggravate the degradation of NP ECM and promote oxidative stress,senescence and apoptosis of NPCs.NF-κB pathway is overactivated in Arg2 over-expressed NPCs,and specific blocking of this pathway could alleviate Arg2 overexpression-mediated oxidative stress,senescence and apoptosis of NPCs.By constructing an animal model of IVDD and using si-Arg2 to interrupt Arg2 expression,the degenerative procedure was remarkably retarded.These results demonstrated that Arg2 modulated oxidative stress,senescence,apoptosis and imbalance of ECM metabolism in the intervertebral discs through the NF-κB pathway,and Arg2 knockdown exerts a protective effects on IVD.