Mechanism Of Bone Marrow Mesenchymal Stem Cells-Derived Exosomes Carrying Circ＿0050205 To Alleviate Intervertebral Disc Degeneration

Background:Intervertebral disc degeneration（IDD）is an age-related,chronic degenerative disease.Studies have shown that circular RNA（circ RNAs）are widespread and abnormally expressed in IDD tissues,which may be involved in the pathological process of IDD,but the specific mechanism remains to be studied.Bone marrow mesenchymal stem cells（BMSCs）have the advantages of easy access,multidirectional differentiation,and strong regenerative capacity,and are the most representative cell source for the study of IDD treatment.Current research indicates that the biological function of stem cells is mainly through paracrine mediated by exosomes,and exosomes can participate in intercellular communication by delivering various active molecules such as non-coding RNAs.The latest research also found that iron metabolism imbalance and ferroptosis are associated with oxidative stress injury of nucleus pulposus cells（NPCs）,which may contribute to the disease progression of IDD.This study aims to investigate whether BMSCs-derived exosomes（BMSCs-Exos）can alleviate IDD progression by regulating the ferroptosis of NPCs,and explore the underlying regulatory mechanism.Methods:1.The mouse IDD model was established by annulus fibrosus puncture,and RNA high-throughput sequencing was performed on the mouse nucleus pulposus（NP）tissue.The mi RNAs target molecules in the downstream of circ＿0050205 were predicted by Circbank and Circinteractome database,and KEGG enrichment analysis was performed.In vitro IDD model was constructed by stimulating NPCs with t BHP.RT-q PCR assays were performed to detect the expression of circ＿0050205,mi R-665 and GPX4 in NP tissues of IDD mice and t BHP-stimulated NPCs.2.NPCs were pretreated with ferroptosis inhibitors Fer-1,Lip-1 and DFO,followed by t BHP stimulation for 6 h or 12 h.CCK-8,Ed U proliferation,flow cytometry were used to detect the proliferation and apoptosis of NPCs,and Western blot was used to detect the expression of MMP13,ADAMTS 5,COL II and Aggrecan proteins.Meanwhile,the expression levels of ROS and Fe²⁺in NPCs were detected.To determine whether ferroptosis is involved in oxidative stress-induced apoptosis and ECM degradation in NPCs.We used BMSC-Exos to treat t BHP-stimulated NPCs and clarified whether BMSC-Exos could inhibit oxidative stress-induced ferroptosis and ECM degradation in NPCs.3.The relationship between circ＿0050205,mi R-665 and GPX4 was verified by dual-luciferase reporter gene,RNA immunoprecipitation（RIP）and RNA pull-down assay.NPCs were transfected with Lv-circ＿0050205,sh-circ＿0050205,mi R-665 mimic,mi R-665inhibitor,Lv-circ＿0050205+mi R-665 minic for 12 h,followed by 50μM t BHP stimulation for 24 hours,respectively,and RT-q PCR to detect expression of related genes.Then,NPCs were treated with Lv-GPX4,BMSC-Exos-Lv-circ＿0050205+sh-NC,BMSC-Exos-Lv-circ＿0050205+sh-GPX4,respectively,and stimulated by t BHP after 12 h.Proliferation and apoptosis of NPCs were detected by CCK-8,Ed U proliferation and flow cytometry.Immunofluorescence assay was used to detect the expression of ECM metabolism-related proteins.Meanwhile,the expression levels of ROS and Fe²⁺in NPCs were detected.To clarify the mechanism of BMSC-Exos in treating oxidative stress injury of NPCs.4.IDD mouse model was used to verify the therapeutic effect of BMSC-Exos in vivo.Forty C57BL/6J mice were randomly divided into 5 groups of 8 mice each,and the experimental groups were as follows:Control,IDD+Saline,IDD+BMSC-Exos+vector,IDD+BMSC-Exos+Lv-circ＿0050205+sh-NC,IDD+BMSC-Exos+Lv-circ＿0050205+sh-GPX4.Lentivirus（10μL 5×10⁹ PFU）was immediately injected into the punctured site.A week later,the mice（except the sham-operated mice）were injected with exosomes（3μg/μL in100μL）via caudal vein once a week for consecutive 6 weeks,and NP tissues were collected for further experiments at 8 weeks postoperatively.The histomorphological changes of the intervertebral discs were identified by imaging and histological examination（HE and Oil red O staining）.RT-q PCR and immunohistochemical assays were performed to detect the expression of relevant genes and ECM metabolism-related proteins in NP tissues.In addition,the expression level of Fe²⁺in NP tissues was detected.Results:1.We screened 337 up-regulated circ RNAs and 283 down-regulated circ RNAs from NP tissues of IDD mice by performing RNA sequencing,among which circ＿0050205 exhibited the largest fold change.Bioinformatics analysis shows that there are four mi RNAs targets（mi R-665,mi R-1248,mi R-1272 and mi R-1265）in the downstream of circ＿0050205.The target gene of mi R-665 is GPX4,which plays an important role in the ferroptosis regulatory network.In oxidative stress-induced NPCs and NP tissues of IDD mice,circ＿0050205 and GPX4 gene expression were decreased,while mi R-665 expression was increased.2.After t BHP stimulation,the activity and proliferation of NPCs decreased and apoptosis increased,and the expression of MMP13 and ADAMTS 5 protein increased,while the expression of COL II and Aggrecan decreased,and the expression levels of ROS and Fe²⁺in NPCs increased.Ferroptosis-specific inhibitors can block the damage of t BHP to NPCs.These results suggested that ferroptosis is involved in oxidative stress-induced apoptosis and ECM degradation in NPCs.BMSC-Exos treatment alleviated oxidative stress-induced ferroptosis and ECM degradation in NPCs.3.The results of dual luciferase reporter gene,RIP and RNA pull-down experiments showed that there were binding sites between circ＿0050205,mi R-665 and GPX4.RT-q PCR experiments revealed that the expression of mi R-665 was reduced after Lv-circ＿0050205transfection of NPCs,and knockdown of circ＿0050205 showed the opposite result.The expression of GPX4 in NPCs was correspondingly decreased and increased after transfection with mi R-665 mimic or mi R-665 inhibitor.Lv-GPX4 transfection reduced apoptosis and ECM degradation,and decreased ROS and Fe²⁺expression levels in NPCs.Treatment with BMSC-Exos or BMSC-Exos-Lv-circ＿0050205 can inhibit ferroptosis and ECM degradation in NPCs,and BMSC-Exos-Lv-circ＿0050205 is more effective than BMSC-Exos.However,BMSC-Exos-Lv-circ＿0050205 could not inhibit ferroptosis and ECM degradation in NPCs transfected with sh-GPX4.The above results suggest that BMSC-Exos can alleviate the damage of oxidative stress on NPCs by regulating the circ＿0050205/mi R-665/GPX4 signaling axis.4.In vivo experimental studies revealed that BMSC-Exos treatment alleviated the destruction of intervertebral disc structure,improved the histomorphology,and also inhibited the degradation of ECM and the imbalance of Fe²⁺expression levels in NP tissues.The protective effect of BMSC-Exos on intervertebral discs disappeared after knockout of GPX4 gene in mice.Conclusions:BMSC-Exos can inhibit ferroptosis and ECM degradation in NPCs by transferring circ＿0050205 and regulating mi R-665/GPX4 signaling axis,thus alleviating IDD progress.Circ＿0050205 may be a potential target for IDD treatment.