The Effects And Mechanisms Of The Oxidative Damage Induced By Medical Ozone On Neurons And The Immune Regulation

BackgroundMedical oxygen-ozone technology shows good prospects in pain clinic recently. However, ozone has toxic effect on cells with its strong oxidation. It is not ozone that contacts with cells of organism, but reactive oxygen species (ROS) and lipid peroxidation products (LOPs). There has a powerful and articulate antioxidant system in the organisms and cells which is able to neutralize ROS and LOPs. Once ROS and LOPs is beyond the capacity of antioxidant systems, ROS and LOPs will induce oxidative damage to the body. The clinical concentration and dose of ozone has not yet formed a unified standard. Our previous work found that the higher concentrations (50,80μg/ml) of ozone could cause the oxidative damage on spinal cord of rabbit,60μg/ml ozone could also lead to the cell membrane damage of neurons in vitro reflecting with the increase of LDH leakage and MDA.The toxicity of ozone leads to a reduction of antioxidizing reserves and creates a cellular redox state disturbance with important consequences concerning cellular functionality. Excessively high levels of ROS could lead to apoptosis which has close relationship with many proteins (i.e., Bcl-2and Bax). The proportion of apoptotic factor Bax and anti-apoptotic factors Bcl-2has an important role in the regulation of cell survival and death. Numerous studies have implicated that the activation of JNK (c-Jun N-terminal kinase)/SAPK (stress-activated protein kinase) plays a critical role in induction of apoptosis in neurons, and ROS could activate upstream kinases directly and inactivate inhibitors indirectly, promoting prolonged JNK activation. JNK regulates the activation of non-nuclear substrates including Bcl-2family members. Bcl-2, antiapoptotic protein, could enhance cell survival. Targeted gene disruption studies indicate that Bax, proapoptotic protein, is essential for JNK-dependent apoptosis. It is possible that the activation of JNK may contribute to ozone mediated apoptosis through regulating the activation of some Bcl-2family members,The body can spontaneously initiate the protective immune response to against the peroxidation injury. The lipid peroxidation activated by oxidative stress may further induces the activation of the immune response. Toll-like receptors (TLRs) play a pivotal role in defense against invading pathogens through detection of pathogen-associated molecular patterns (PAMPs). TLRS can activate innate immunity and regulate acquired immunity. TLR4recognizes lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria and LPS could induce secretion of proinflammatory cytokines such as TNF-α and IL-6. Appropriate activation of TLRs is beneficial to the body against pathogens infection, while the persistent immune response induced by excessive activation of TLRs can bring adverse effect to the body. Although activation of TLR signaling and secretion of proinflammatory cytokines are important for the elimination of invading microorganisms, uncontrolled activation may lead to autoimmune and inflammatory diseases. Therefore, TLR signaling must be tightly controlled to prevent excessive inflammatory response.Ubiquitin is one form of the post-translational modifications protein modification after translation, which regulates various biology process of cell. Ubiquitination is one of the major mechanisms of inhibit or termination of signal protein which is regulated by negative regulatory factors. The protein labeled by ubiquitin was degradated after specific recognition, and this process occupies an important position in the antigen-presenting and inflammatory response of cells. Thus, ubiquitin-protein ligase E3has a central role in ubiquitination. Studies have shown that the WW domain E3ubiquitin-protein ligase WWP1may be involved in the process of the body’s inflammatory response, the generation of WWP1was induced by the inflammatory cytokines TNF-α. The LPS-TLR4mediated inflammatory reaction can active the innate immune response, while WWP1regulate the innate immunity of the body by antagonizing the DAF-2insulin signal transduction pathways. The over-expression of WWP1caused by TGF-β may be related with the negative feedback regulation of the inhibition of WWP1to TGF-β.The balance between anti-inflammatory reaction mediated by TGF-β and inflammatory reaction mediated by LPS-TLR4plays an important role in the occurrence of many inflammatory diseases.Our previous studies strongly suggested that the oxidative stress induced by ozone could injure nerve system, which was also reported by clinical research recently. Although it has been reported that ozone has oxidative toxicity to biological tissues, there is no evidences to show neurotoxity of ozone and the mechanisms of this modulation are unclear either. WWP1regulates a variety of cellular biological processes including protein trafficking and degradation, signaling, transcription, and viral budding, has also been implicated in several diseases, such as cancers, infectious diseases, neurological diseases, and aging. However, it is still unclear whether WWP1plays an important role in TLR-mediated inflammation as E3ubiquitin ligase.ObjectiveThe present study was designed to investigate the effects of different concentrations (20,40,60μg/ml) of ozone on the expression of JNK/MAPK signal protein of neurons, and to clarify the the role of JNK/MAPK signaling pathway in the mechanisms of the oxidative damage injured by medical ozone. The study was also designed to investigate the effects of WWP1on the expression of signal protein and inflammatory cytokines induced by LPS and the regulation of WWP1on the LPS-TLR4pathways, and to clarify the underlying mechanisms. It will provide the new target of the negative regulation on the inflammatory response.Methods1. Toxic effect of different concentrations (20,40,60μg/ml) of ozone on neuronsThe spinal neuron was identified by immunofluorescence. The morphological changeswere observed using Hoechst33258staining method. Whole cell patch clamp technique were used to observed the effects of the lower concentration (15,20μg/ml) of ozone on spinal neurons. The cell viability was tested by MTT assay and the apoptosis ratio was analyzed by flow cytometry. The expression of Bax and Bcl-2was detected by Western blotting and RT-PCR.2. Effects of ozone (20,40,60μg/ml) on the expression of MAPK signaling pathway The expression of JNK, p-JNK, ERK and p-ERK was detected by western blotting. 3. Effect of AS601245on ozone-induced apoptosisThe spinal neurons and PC12cells were pretreated with AS601245(0.1μM) for40min, followed by co-treatment with different concentrations (20,40,60μg/ml) of ozone for12h. The expression of JNK, p-JNK, ERK and p-ERK was detected by Western blotting,.the effects of AS601245on cell viability were evaluated by MTT assay. The apoptosis ratio of cells was quantitative analyzed by flow cytometry. The expression of Bax and Bcl-2was detected by Western blotting and RT-PCR.4. The role of JNK pathway in the oxidative damage induced by ozone in vivoThe rats were injected with different concentrations of ozone (20,40,60μg/ml) for24h via the implanted intrathecal catheters. The BBB Locomotor Score was used to assessment the degree of spinal injury. The expression of Bax and Bcl-2was detected by Western blotting and RT-PCR. The expression of JNK, p-JNK, ERK and p-ERK was detected by western blotting.The rats were pretreated with AS601245(4.5mg/kg) via tail vein for40min, followed by intrathecal injection with different concentrations (20,40,60μg/ml) of ozone for24h. The expression of JNK, p-JNK, ERK and p-ERK was detected by Western blotting. The expression of Bax and Bcl-2was detected by Western blotting and RT-PCR.5. Effects of WWP1on the immune response IL-6induced by LPS or TNF-aThe WWP1in peritoneal macrophages/RAW264.7was silenced/overexpresses by SiRNA/Plasmid transfections, the expressions of TNF-a and IL-6induced by LPS or TNF-a respectively were tested by ELISA and RT-PCR, the protein expression of IRF3, NF-κB and MAPKs (JNK, ERK and P38) by western blotting.6. The possible relation between WWP1and LPS-TLR4-MyD88His-tagged WWP1was co-expressed with myc-tagged TRAF6and myc-tagged IRAK1in HEK293T cells, Immunoprecipitation and Immunoblot analysis was used to detect the relation between WWP1and TRAF6, IRAK1. The expression of TRAF6and IRAK1was detected using Western blotting with the attendance of WWP1, which was detected again after adding the protease inhibitor MG132.The WWP1in peritoneal macrophages/RAW264.7were silenced/overexpresses by SiRNA/Plasmid transfections, Western blotting was to test the expressions of TRAF6and IRAK1and investigate that knockdown of WWP1reduces LPS-induced K48or K63-linked poly-ubiquitination of endogenous TRAF6.7. The possible mechanism of WWP1on the proteasomal degradation of TRAF6The WWP1in C5/B7rats was knockdown by SiRNA transfections, ELISA was used to detect TNF-a, IL-6secretion in serum when the tested mice were given intraperitoneal injection of PBS or LPS. Then endotoxin-shock mouse model was established by intraperitoneal injection of LPS, the lungs injure were detected using Hematoxylin and Eosin staining method.Results1. The toxic effect of different concentrations of ozone in neurons and the underling mechanismNormal neurons could still be seen in Groups O20(20μg/ml) and C (0μg/ml), but rare cells survived in Group O60(60μg/ml). Cell neurites became twitched or broken, forming rosary-like appearances in groups of higher ozone concentration (60μg/ml). Nucleus of neuron was stained pallid blue with even distribution by Hoechst33258. Nucleus were stained brighter and condensed in all ozone exposure groups, and this effect was concentration-correlated. Peak calcium current density was significantly increased in Group O15and Group O20groups compared with that in the control group (P<0.05). Higher concentrations of ozone (40,60μg/ml) decreased the cell viability (P<0.01) and increased the cell apoptosis (.P<0.01). The level of p-JNK but not p-ERK was significantly enhanced after the injection of ozone, and the expression of Bax (P<0.01) but not Bcl-2was also enhanced significantly after the injection of ozone. But the inducible protein and mRNA expression of Bax (P<0.01) and the apoptosis induced by ozone (P<0.05) was inhibited in the AS601245group.2. The toxic effect of different concentrations of ozone in rats and the underling mechanismCompared to the control group, injecting ozone could significantly impact the rats’ locomotor behavior, especially at60μg/ml and the point of6h (P<0.01). The treatment of AS601245could mitigate this injure. Ozone could induce the phosphorylation of SAPK/JNK and expression of Bax, but the inducible expression of Bax was suppressed by AS601245(P<0.01). 3. Knockdown of WWP1enhances TNF-aand IL-6production induced by LPS but not TNF-aThe expression of TNF-aand IL-6was increased in theperitoneal macrophages of Lesh WWP1+LPS group compared with the NC groups and Lesh WWP1+TNF-a group (P<0.01), which was decreased in RAW264.7cells of His-WWP1+LPS group compared with His-Control,His-C890A groups and His-WWP1+TNF-a group (P<0.01).In vivo studies, compared to the Lesh NC+LPS group, TNF-a, IL-6secretion in serum was increased in Lesh WWP1+LPS group (P<0.01), Histological analysis of lung tissue showed more inflammation in the lungs of Lesh WWP1mice than in the lungs of Lesh NC mice when the Lesh NC, LeshWWPl mice were challenged with intraperitoneal LPS for48h.4. WWP1inhibits IκB-μ, NF-κB and MAPK Activation stimulated by LPSCompared to the Lesh NC group, the expression of p-IκB-a, p-65, p-JNK, p-ERK, p-P38all increased, especially at the90min, but there was no significant differences of the IRF3phosphorylation between groups. Compared with the His-Control and His-C890A groups, the expression of p-IκB-α, p-65, p-JNK, p-ERK, p-P38but not IRF3decreased significantly.5. WWP1interacts with TRAF6but not IRAK1both in vitro and in vivoImmunoprecipitation and Immunoblot analysis showed that His-WWP1could associate with the myc-tagged TRAF6, but not myc-tagged IRAK1, both the wide type WWP1protein and WWP1-C890A mutant protein could interact with TRAF6normally in vitro, WWP1was weakly associated with TRAF6in unstimulated RAW264.7cells, and this association ability increased after stimulation with LPS in30min and60min. Thus WWP1promoted degradation of TRAF6, and this degradation disappeared when we added MG132, knockdown of WWP1could increase the endogenous expression of TRAF6but not IRAK1, and endogenous TRAF6but not IRAK1in His-WWP1cell line was much lower than in His-Control and His-C890A cell line. 6. Knockdown of WWP1reduces LPS-induced K48but not K63-linked polyubiquitination of endogenous TRAF6The Immunoprecipitation experiments indicated that TRAF6was polyubiquited both in K48and K63-linked polyubiquitination way when cells were stimulated by LPS, and knockdown of WWP1reduced LPS-induced TRAF6polyubiquitination, knockdown of WWP1reduced LPS-induced K48-linked but not K63-linked polyubiquitination of TRAF6.Conclusion1. Medical ozone induces oxidative damage on neurons with a concentration-dependent manner.2. JNK inhibitor pretreatment can significantly reduce the apoptosis and oxidative damage caused by ozone and improves motor function score.3. Ozone induced neurotoxicity is dependent upon generation of apoptosis mediated by activation of J NK signaling pathway.4. WWP1inhibits TNF-α and IL-6production induced by LPS5. WWP1reduces LPS-induced K48-linked polyubiquitination of endogenous TRAF66. WWP1negatively regulates TLR4-Mediated TNF-αand IL-6production by proteasomal degradation of TRAF6.