Propionibacterium Acnes Induces Pyroptosis Of Degenerated Nucleus Pulposus Cells Via NLRP3-dependent Pathway And Its Mechanism

Intervertebral disc degeneration(IVD)is one of the most important causes of chronic low back pain(LBP).With the development of medicine,the increasement of average life expectancy and the accelerating of the aging society,the incidence of IVD is also increasing year by year and causes serious family and socio-economic burdens.The specific mechanism of IVD is still controversial at present,so there is lack of effective treatment.Recently,more and more researchers have found that infection plays an important role in the process of IVD,of which Propionibacterium acnes(P.acnes)is the most widely studied.However,the effect of pyroptosis of human degenerated nucleus pulposus cells induced by P.acnes and its underlying mechanism lack systematic research.Our project is intended to be elaborated from the following four aspects: 1.Extraction of human nucleus pulposus tissue and cells and the effect of P.acnes infection on the secretion of inflammatory cytokines in nucleus pulposus cells;2.P.acnes induces activation of pyroptosis of human nucleus pulposus cells;3.MCC950 inhibits inflammation and alleviatespyroptosis through TXNIP-NLRP3 axis pathway;4.The influence of MCC950 on P.acnes infection in lumbar intervertebral disc.Part1.Extraction of human NP tissue and cells and the effect of P.acnes infection on the secretion of inflammatory cytokines in NPCsObjective:To investigate whether P.acnes can induce the release of inflammatory cytokines in NP tissue and cells.Methods : The inclusion and exclusion criteria of specimens were established.Preoperative imaging data of patients were collected and corresponding questionnaire scores were made.After collecting normal and degenerative NP tissues,immunohistochemistry was used to detect the expression of NLRP3 inflammasome.Moreover,part of the NP tissue samples was collected for anaerobic culture and 16 S r DNA was used to identify whether there is existence of P.acnes.Primary NPCs were isolated and extracted by enzyme sequential digestion.P3 generation NPCs were used for subsequent experiments.After co-culturing NPCs with P.acnes of different multiplicity of infection(MOI)for various times,western blot and ELISA were used to detect the expressions of IL-1β and IL-18,so as to screen out the best concentration and treatment time of P.acnes.Results : P.acnes were detected in NP tissue specimens of some patients in LDH group.Patients with P.acnes infection had significantly worse results of preoperative imaging examination and questionnaire scores.Immunohistochemical results showed that NLRP3 staining in P.acnes infection group was strongly positive,indicating the aggravation of IVD induced by P.acnes.After co-culturing NPCs with P.acnes,Western blot and ELISA results proved that P.acnes could induce the expressions of IL-1β and IL-18 in NPCs.The best concentration and treatment time of P.acnes is MOI 2.0 treatment for 24 h.Conclusion : P.acnes could induce the release of inflammatory cytokines in NP tissues and cells.PART2.P.ACNES INDUCE THE ACTIVATION OF PYROPTOSIS OF HUMAN NPCSObjective:To investigate whether P.acnes can induce the activation of pyroptosis of human NPCs.Methods:Primary NPCs were collected from patients in LVF group and P3 generation NPCs were used for subsequent experiments.NPCs treated with P.acnes of MOI 2.0 was served as experimental group,while17% FBS culture condition was served as control group.Extracting proteins after 24 h,western blot was used to detect the expressions of Caspase-1 and Caspase-5.To identify whether Caspase-1 or/and Caspase-5participated in the secretion of IL-1β in NPCs induced by P.acnes,inhibitors of both(Z-YVAD-FMK for Caspase-1,Z-WED-FMK for Caspase-5)were used to pretreat NPCs before co-culture and then ELISA was used to detect the expression of IL-1β.After that,to identify whether pyroptosis was activated in NPCs stimulated by P.acnes,western blot was used to detect the expressions of pyroptosis-related protein(NLRP3,ASC,Pro-caspase-1,Full length GSDMD 和 Cleaved GSDMD).Results:The western blot results showed that P.acnes could induce the expressions of Caspase-1 and Caspase-5 in normal NPCs,while the ELISA results exhibited that only after the pretreatment of Caspase-1inhibitor,the expression of IL-1β was inhibited in a concentration-dependent manner.Comparing with the control group,the western blot results showed that pyroptosis-related proteins were detected in normal NPCs stimulated by P.acnes.Conclusion : P.acnes could include the activation of pyroptosis in human NPCs.PART3.MCC950 INHIBITS INFLAMMATION AND ALLEVIATES PYROPTOSIS THROUGH TXNIP-NLRP3 AXIS PATHWAYObjective:To investigate whether MCC950 can alleviate pyroptosis by inhibiting inflammation through TXNIP-NLRP3 axis pathway.Methods:Primary NPCs were collected from patients in LDH group and P3 generation NPCs were used for subsequent experiments.Before P.acnes stimulation,NPCs were pretreated with different concentrations of MCC950 and then ELISA was used to detect the expression of IL-1β to screen out the best concentration of MCC950.NPCs were divided into four groups: Control group,Control+MCC950 group,P.acnes group and P.acnes+MCC950 group.Western blot was used to detect the expressions of pyroptosis-related proteins(NLRP3,Pro-Caspase-1,ASC,Cleaved caspase-1,Full length GSDMD and Cleaved GSDMD)and inflammatory cytokines(IL-1β and IL-18)in each group.PI staining was used to compare the rates of pyroptosis among Control group,P.acnes group and P.acnes+MCC950 group.In order to further investigate the underlying mechanism of MCC950 in inhibiting pyroptosis,TXNIP protein expression was detected by western blot.Subsequently,TXNIP was knocked down by si RNA and western blot was used to confirm its success.Then NPCs were divided into four groups: Control group,Control+si RNA group,P.acnes group and P.acnes+si RNA group.To investigate the role of TXNIP in the process of MCC950 in inhibiting pyroptosis,western blot was used to detect the expressions of NLRP3,TXNIP,IL-1β and Cleaved Caspase-1.Results:The ELISA results showed that the secretion of IL-1β was inhibited in a concentration-dependent manner after the pretreatment with MCC950.When MCC950 concentration is at 1.0 μM,the inhibition of IL-1β is most significant.So the best concentration of MCC950 is at 1.0μM.The western blot results showed that the expressions of pyroptosis-related proteins(NLRP3,Cleaved Caspase-1,Cleaved GSDMD)and inflammatory cytokines(IL-1β and IL-18)were inhibited by the pretreatment of MCC950.Moreover,the PI staining results also show the effect of MCC950 in inhibiting pyroptosis.During the process of exploring the specific mechanism of MCC950,the western blot results showed that P.acnes could upregulate the TXNIP expression,while MCC950 could reverse this result.When TXNIP was knocked down by si RNA,the western blot results showed the expressions of NLRP3,IL-1β and Cleaved Caspase-1 could be inhibited.Conclusion : MCC950 could alleviate pyroptosis by inhibiting inflammation through TXNIP-NLRP3 axis pathway.PART4.THE EFFECT OF MCC950 ON P.ACNES INFECTION IN LUMBAR INTERVERTEBRAL DISCObjective:To investigate the effect of MCC950 on P.acnes infection in lumbar intervertebral disc in animal experiments.Methods : Twelve New Zealand rabbits were purchased from the animal center of Chongqing Medical University.The rabbits were divided into two groups at random: group A and group B.According to our research group previous experience,we chose fiber ring puncture through the abdomen.5.0μL of P.acnes was injected into the L3-L4 disc,and L4-L5 disc was injected with 2.5μL P.acnes and 2.5μL MCC950 and L5/6 disc was injected with 5.0μL normal saline as control in the group A.In the group B,5.0ul of normal saline was injected into L3-L4,L4-L5 and L5-L6 discs as control group.The MRI images were obtained at 2/4/6/8 weeks after operation.After euthanizing the animals,H&E staining,Alcian blue staining and immunohistochemistry(Aggrecan and COL-Ⅱ)were used to check the degree of intervertebral disc degeneration.Results:According to MRI imaging results,P.acnes+MCC950 group had a significantly higher MRI index compared with that in only P.acnes infection group,which proved that MCC950 could ameliorate IVDD accentuated by P.acnes in vivo.H&E staining showed that not only did the NPCs clump together,but also the number is obviously higher in P.acnes+MCC950 group comparing with P.acnes group(P<0.05).Alcian blue staining also showed that the OD value of NPCs was higher in P.acnes+MCC950 group than that in P.acnes group(P<0.05).Besides,in immunohistochemical results,the expressions of aggrecan and COL-Ⅱwere higher in P.acnes+MCC950 group than that in P.acnes group(P<0.05).Conclusion : MCC950 could alleviate the process of intervertebral disc degeneration induced by P.acnes infection.