| Objective:The kallikrein-kinin system(KKS)plays an important role in maintaining tissue homeostasis,kidney function,and blood pressure.A recent study has shown that kininogen(KNG)is highly expressed in the nucleus pulposus cells(NPCs)of aged rats.However,it is not yet known whether the key components of KKS,such as the bradykinin 1/2 receptors(B1R/B2R),are present in human NPCs,or what role bradykinin(BK)plays in these cells.The aim of this study is to confirm the existence of KKS in the nucleus pulposus of human intervertebral discs,and to explore the role of BK in oxidative stress-induced nucleus pulposus cell death and intervertebral disc degeneration(IVDD).The study aims to elucidate the partial role of KKS in the process of IVDD,and to lay an experimental foundation for targeted KKS therapy to delay IVDD in clinical practice.Method:1.Study on the correlation between KKS and IVDD.We collected 10 clinical nucleus pulposus tissue specimens based on the Pfirmann IVDD grading criteria,including 5 cases of mild degeneration(grades I and II)and 5 cases of severe degeneration(grades III to V).Pathological methods were used to observe the cellular and tissue characteristics of the tissue specimens with mild and severe degeneration.Western blotting(WB)was employed to detect the molecular expression differences of the nucleus pulposus at different degeneration grades.Additionally,immunohistochemistry and WB methods were used to validate the presence of the KKS(including KNG,B1 R,and B2R)in human nucleus pulposus tissue and determine its expression in nucleus pulposus tissue of different degeneration grades.Human and rat primary nucleus pulposus cells(NPCs)were isolated and extracted for the study.The primary NPCs from both humans and rats were intervened with important biological factors(IL-1β,TNF-α,and TBHP)known to induce nucleus pulposus degeneration.PCR,WB,and immunofluorescence techniques were employed to confirm the presence of KKS in NPCs and its correlation with NPC degeneration.2.Study on the role and mechanism of BK in NPCs after TBHP treatment.We cultured primary rat NPCs and divided them into experimental groups:Control group,BK group,TBHP group,and BK+TBHP group.Various methods such as CCK-8 assay,DCFH-DA fluorescence probe,JC-1 mitochondrial membrane potential detection,Hoechst 33258 staining,β-galactosidase senescence staining,flow cytometry,immunofluorescence,and Western blotting were employed to investigate the effects of BK on TBHP-induced oxidative stress,apoptosis,senescence,extracellular matrix metabolism,and the PI3K/AKT pathway in NPCs.Furthermore,the experimental groups were divided into Control group,TBHP group,BK+TBHP group,HOE140+BK+TBHP group,and LY294002+BK+TBHP group.The aforementioned methods were used to verify whether BK protected cells from TBHP-induced apoptosis,senescence,and extracellular matrix degradation through the activation of the B2R/PI3K/AKT pathway.3.Orthogonal design optimization of preparation process for BK/PLGA sustained-release microspheres and in vitro drug release study.The microspheres were prepared by the double emulsion solvent evaporation method,with drug loading and encapsulation efficiency of BK in microspheres as the main evaluation indicators.Four process conditions,including PLGA solution concentration,internal aqueous phase BK drug concentration,external aqueous phase PVA concentration,and stirring speed,were studied using a four-factor,three-level orthogonal experiment design.The optimized microspheres were characterized,and the drug release in vitro was determined.Co-cultivation with primary rat NPCs was performed to evaluate the effect on cell viability.4.Animal experimental study on the delaying effect of BK/PLGA microspheres on IVDD.We constructed a rat tail IVDD model using the needle puncture method and injected BK/PLGA microspheres to evaluate the therapeutic or delaying effect of BK on IVDD in rats.The methods employed are as follows: 27 Sprague-Dawley(SD)rats were randomly divided into 3 groups: sham surgery group(Sham),IVDD group,and IVDD + BK/PLGA microspheres group(IVDD + BK Microspheres),with 9 rats in each group.The appearance of the tail puncture wounds and general behavior of the rats were closely monitored after surgery.On the day of surgery,as well as at 1 and 2months post-surgery,3 rats were randomly selected from each group and euthanized for X-ray,MRI imaging,and histopathological examination to observe the therapeutic effects of BK/PLGA microspheres on degenerated intervertebral disc nucleus pulposus.Result:1.DDiscovery of KKS in human nucleus pulposus tissue and human/rat NPCs,and its association with IVDD.The results of this study demonstrated the expression of extracellular matrix,senescence,and apoptosis markers in nucleus pulposus tissue through Western blotting(WB),validating the degree of degeneration in the collected tissue samples at the molecular level.Immunohistochemistry confirmed the presence of key components of the kallikrein-kinin system(KKS),namely KNG-1,B1 R,and B2 R,in the nucleus pulposus tissue,with their high expression observed in degenerated NP tissue.PCR and WB analysis further confirmed the presence of KNG-1,B1 R,and B2 R in NPCs,with their expression levels increased after intervention,showing correlation with degeneration.2.BK activates the B2R/PI3K/AKT pathway to protect cells from apoptosis,senescence,and extracellular matrix degradation induced by TBHP.The results of the study showed that BK exhibited a dose-dependent increase in the viability of rat NPCs and reduced the production of reactive oxygen species(ROS)in normal NPCs.Pre-treatment with BK significantly decreased the elevated ROS levels induced by TBHP,restored the reduced mitochondrial membrane potential,increased the functionality of mitochondria by upregulating the protective protein Bcl-2,and rescued NPCs from TBHP-induced apoptosis.Moreover,the cellular senescence staining results revealed that BK alleviated the senescence of TBHP-induced NPCs,and the levels of senescence markers(P53,P21,and P16)were reduced in the BK intervention group.Additionally,BK promoted increased synthesis of the extracellular matrix,as evidenced by elevated expression of proteoglycan(Aggrecan)and type II collagen(Collagen-II),while reducing the expression of extracellular matrix degradation markers,such as matrix metalloproteinase-3(MMP-3)and matrix metalloproteinase-9(MMP-9).After inhibiting the B2 R and PI3K/AKT pathways,BK cannot reduce apoptosis,alleviate senescence,and promote extracellular matrix synthesis.3.The in vitro drug release of BK/PLGA sustained-release microspheres was stable,and they exhibited excellent biocompatibility.BK/PLGA sustained-release microspheres were prepared by the double emulsion solvent evaporation method.Orthogonal experimental results showed that the optimal conditions for preparing microspheres were PLGA input of 100 mg,BK input of0.5mg,PVA concentration of 1.5%,and stirring speed of 400r/min.Under these conditions,microspheres with a regular morphology,a particle size of approximately80μm,an encapsulation efficiency of 74.10±9.17%,and a drug loading rate of0.370±0.046% were prepared,with sustained release of BK for more than 28 days.Co-culture of the microspheres with primary rat NPCs in vitro showed that the BK/PLGA microspheres had no significant toxicity,could improve cell viability,and had a slight proliferative effect.4.BK/PLGA microspheres delayed the progression of IVDD in rats after needle puncture.By observing the postoperative time points at day 0,1 month,and 2 months using methods such as molybdenum target X-ray,MRI,and histopathological sections,the results showed that the IVDD group exhibited the disappearance of nucleus pulposus hydration,a decrease in intervertebral height,and disc degeneration after 1month.In contrast,the IVDD+BK Microspheres group maintained a certain level of nucleus pulposus hydration and delayed intervertebral disc degeneration at the1-month and 2-month time points.Similarly,histopathological sections and immunohistochemistry of the tail vertebrae specimens confirmed the targeted and sustained release of BK/PLGA microspheres in the intervertebral disc,precise modulation of extracellular matrix(ECM)metabolism imbalance in IVDD,stabilization of ECM,restoration of tissue function,promotion of nucleus pulposus regeneration,and delayed degeneration of the rat tail intervertebral disc.Conclusion:Our study for the first time discovered the presence of the KKS in human intervertebral disc tissue and NPCs,and its involvement in intervertebral disc degeneration;demonstrating the beneficial protective effect of BK partially activating the bradykinin B2 receptor and downstream PI3K/Akt pathway on NPCs under pathological conditions.KKS may play an important role in intervertebral disc degeneration.BK/PLGA sustained-release microspheres constructed targeting the bradykinin B2 receptor have great potential for treating intervertebral disc degeneration and promoting intervertebral disc regeneration. |
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