The Role And Mechanism Study Of Resveratrol On Nucleus Pulposus Cell Induced By Oxidative Stress

Intervertebral disc degenerative disease(IVDD)is an important age-related disease,and its incidence and morbidity are increasing in the aging society,which has led to a heavy medical burden and social health problem.Intervertebral disc degeneration is an important pathogenesis of IVDD,and oxidative stress-induced nucleus pulposus(NPCs)injury is one of the major pathologic features.Resveratrol(RES)is a natural antioxidant,and previous research group has reported that resveratrol can protect NPCs from apoptosis,but its specific mechanism remains unclear.In addition,RES is poor in water solubility and unstable in vivo,so designing an ideal drug carrier to enable it to be efficiently and stably released in tissues can contribute to the clinical application of RES.In summary,this project intends to solve two major scientific issues:(1)Mechanism of action of RES on NPCs induced by oxidative stress;(2)Design and construction of appropriate RES drug carriers to improve the efficacy of retardation of intervertebral disc degeneration in vivo.SECTION ONE RES PROTECTES AGAINST MITOCHONDRIA DAMAGE INDUCED BY OXIDATIVE STRESSObjective To investigate the effect of RES on the mitochondrial function of NPCs treated with H2O2.Methods Human primary NPCs were isolated and cultured.The third generation cells were treated with different concentrations of H2O2(0,10,20,50,100,and 200 ?mol/l)for 24 h.,then the cell viability was determined by CCK-8.After treatment with 100 ?mol/l H2O2,DCFH-DA fluorescence probe was used to detect intracellular ROS expression,JC-1 staining was used to detect mitochondrial membrane potential,ATP detection kit was used to measure intracellular ATP synthesis,and the effect of H2O2 on mitochondrial damage was evaluated.Annexin V/PI flow cytometry was used to detect apoptosis.To further observe the effects of ROS on the mitochondrial function of NPCs under oxidative stress,50 mmol/1 RES was used to pretreat NPCs for 24 h,and then mitochondrial function and apoptosis were detected.Results CCK-8 results showed that compared with the control group,the relative activities of cells after different concentrations of H2O2 treatment decreased to 92.1%,85.3%,74.2%,48.4%,and 33.9%,respectively,so it can be seen that 100 ?mol/l H2O2 has obvious cytotoxicity to NPCs.After treated with 100 ?mol/1 H2O2,the mitochondrial membrane potential and ATP synthesis of NPCs were significantly decreased and ROS expression was significantly increased(P<0.05),suggesting that H2O2 can cause mitochondrial damage in NPCs.Flow cytometry results further suggested that H2O2 significantly increased the apoptosis rate of NPCs(P<0.05).The RES pretreatment significantly improved the mitochondrial function and apoptosis rate of NPCs(P<0.05).Conclusion Resveratrol can reduce mitochondrial damage induced by oxidative stress and protect cell apoptosis.SECTION TWO RES PROTECTES AGAINST MITOCHONDRIA DAMAGE THROUGH AUTOPHAGY ACTIVATION IN NPCSObjective To explore the mechanisms of RES protecting mitochondrial damage in NPCs under oxidative stress.Methods NPCs were treated with 100 ?mol/1 H2O2 and 100 ?mol/l H2O2 plus 50 mmol/1 RES,respectively.Western blot was used to detect autophagy-related proteins LC3 and P62.To further clarify the regulatory mechanism of RES on autophagy,the addition of bafilomycin A1 followed by western blot detection.After addition of autophagy inhibitor 3-MA,the mitochondrial function and apoptosis of NPCs were measured.Results Western blot results showed that the expression of LC3-II/LC3-I protein could be significantly decreased after H2O2 treatment,but the expression of P62 was increased(P<0.05),suggesting that H2O2 could inhibit the autophagic expression of NPCs.after pretreatment with RES,this trend was reversed and the LC3-II/LC3-I protein expression ratio increased but decreased P62 expression significantly.After BAF treatment,western blot results showed that both LC3-II/LC3-I expression ratio and P62 protein expression were significantly increased(P<0.05),which proved that RES activated the autophagic flow burst of NPCs rather than hindered autophagy degradation.After the addition of autophagy inhibitor 3-MA,the mitochondrial function and apoptosis protection mediated by RES was significantly reversed(P<0.05).Conclusion RES protects mitochondrial damage of NPCs induced by oxidative stress through activation of autophagy and protects cell apoptosis.SECTION THREE SYNTHESIS OF RES-LOADED TARGETING NANOBUBBLESObjective To construct a new type of RES nano-bubbles.Methods PLGA nano-ultrasonic microbubbles containing RES were constructed by double emulsification method and then the CDH2 antibody was conjugated to the surface of microbubbles by carbodiimide method.In vitro microbubble physical properties and targeting of NPCs were detected.Results The RES/AbCDH2 ultrasound microbubbles were synthesized successfully with a diameter of 567.3±114.7 nm and the entrapment efficiency of RES was(50.42±5.16)%.Combined with low intensity focused ultrasound irradiation,the release rate of RES was much higher than that of drug-loaded microbubbles that were not irradiated by ultrasound at the same time point,and the difference was statistically significant(P<0.05).In vitro cytology showed that the microbubbles with CDH2 target had better targeting ability to NPCs,and confocal laser scanning showed that the number of microbubbles accumulated around NPCs was significantly higher than that without CDH2(P<0.05).After combined treatment with RES/AbCDH2 group,the expression of SIRT1,extracellular matrix type II collagen and Aggrecan were significantly increased,and apoptosis was significantly inhibited,suggesting a good anti-catabolic effect.Conclusion RES/AbCDH2 ultrasound nano-microbubbles showed good targeting and anti-catabolic effects in vitro cytology experiments.SECTION FOUR RES-LOADED NANOBUBBLES FOR IN VIVO STUDYObjective To investigate the effect of RES/AbCDH2 ultrasound microbubbles on delaying degeneration of intervertebral discs.Methods The rabbit degeneration model was established by acupuncture.The RES/AbCDH2 ultrasound microbubbles were injected into the surgical disc via local injection,supplemented with ultrasound irradiation.Eight weeks after operation,magnetic resonance was used to evaluate the degree of disc degeneration.Animals were sacrificed and tissue samples were taken for aliblue staining and SIRT1 immunohistochemistry.Results Rabbit IDD animal model was successfully constructed.After RES/AbCDH2 microbubbles combined with ultrasound irradiation,MRI showed a significant delay in disc degeneration at 8 weeks compared with other control groups;Histological examination suggested that in RES/AbCDH2 microbubbles group the content of extracellular matrix was more and the expression of SIRT1 were increased and the difference was statistically significant(P<0.05).Conclusion The application of RES/AbCDH2 ultrasound microbubbles can significantly delay the degeneration of intervertebral discs,demonstrating a good clinical application prospect.