| BackroundOsteoarthritis(OA)is characterized by joint swelling,chronic pain and limited mobility,which principal pathological changes were as articular cartilage degeneration and chronic inflammation.The main causes of degeneration of articular cartilage are aging of chondrocytes,increased degradation of extracellular matrix,and osteochondral bone formation leading to osteophyte formation.With the development of osteoarthritis,peripheral nociceptors are activated by involved joints and synovitis,and nociceptive signals are transmitted to the spinal cord,resulting in increased excitability of spinal cord injury neurons and changing the the mechanism of pain,"which progress from nociceptive the feeling of progression to neuropathic pain(NP).OA brings mental and physical pain to patients,and it also puts a serious challenge for clinicians.Therefore,exploring the mechanism of OA and the effective methods of treating OA have become hot topics in clinical research.Medical ozone originated at the end of the 19th century,and Wolff reported that ozone was used in combat wound treatment,which exerts anti-inflammatory and bactericidal effects.Later,Dr.Bocci V of Italy reported that medical ozone has the effect of anti-inflammatory,analgesic and promoting local tissue regeneration.Medical ozone has become widely utilized due to several advantages in clinical practice,such as uncomplicated operation,low cost and small trauma.Medical ozone is an unstable blue gas,which is an isomer of oxygen.Its water solubility is 10 times higher than that of oxygen,20 times higher than that of air,and it is easy to dissolve in the blood and tissue fluid.Ozone rapidly decomposes into oxygen and oxidizing oxygen atoms at room temperature,and the latter reacts with biomolecules to produce reactive oxygen species(ROS)and lipid hydroperoxides(LOPs),which can be used to treat infection of oral,skin and mucosal.In the clinical pain management,medical ozone exerts anti-inflammatory,anti-oxidative and analgesic effects,and it is efficacious in the treatment of lumbar disc herniation(LDH),soft tissue pain,OA and so on,which is recognized by doctors and patients.It has been found that intra-articular injection of low concentration of medical ozone(<30 ?g/mL)can inhibit the production of IL-1? and TNF-?,reduce the degradation of the extracellular matrix of chondrocytes,and protect chondrocytes.At present,there are few basic researches on the mechanism of OA treatment and analgesia mechanism by medical ozone,which limits the clinical normative and safe application of medical ozone.The current study found that the main cause of cartilage degeneration in OA is the decreased autophagy of chondrocytes.Maintaining a certain level of autophagy in chondrocytes can prevent the cartilage degeneration,which is an important way for chondrocytes to survive in OA.Therefore,increasing autophagy of chondrocytes has become a therapeutic target for OA.Autophagy is an essential way to maintain cell homeostasis and survival.The level of autophagy increases to maintain cell survival when cells are stressed,such as energy deficiency,oxidative stress and hypoxic environment.The autophage-related gene(ATG)was first discovered in yeast,and the corresponding eukaryotic genes were also identified.Among them,ATG1,ATG6 and ATG8 correspond to mammalian Unc-51-like kinase 1(ULK-1),Beclinl and microtubule-associated protein 1 light chain 3(LC3),respectively.LC3 is a key gene of autophagy,and the ubiquitinated binding protein SQSTM1/p62(p62)is also involved in the occurrence of autophagy.LC3 is widely expressed in soluble proteins of mammalian tissues and cultured cells,which is called cytosolic LC3(LC31).In the autophagy initiation phase,LC3? is converted to LC3? in the form of a phospholipid conjugate compound.ULK-1 is essential for the formation of autophagic vacuoles,and the inactivation of ULK-1 kinase inhibits the formation of LC3?.ULK-1 is negative controlled by the mammalian target of rapamycin(mTOR),which is one of key regulators of autophagy.mTOR is inhibited in the absence of energy,and then ULK-1 activity is enhanced due to the inhibition of mTOR,which up-regulates autophagy.Beclinl gene is also called BECN1 gene.The expression of Beclinl is positively correlated with the level of autophagy and is a positive regulator of autophagy.mTOR can be regulated by multiple signaling pathways,of which AMPK/mTOR signaling pathway is widely concerned.AMPK negatively regulates mTOR to regulate autophagy.It has been demonstrated that oxidized lipoprotein a enhances autophagy in vascular endothelial cells through the AMPK/mTOR signaling pathway,and AMPK activation is reactive Oxygen Species(ROS)-dependent.Current research suggests that AMPK can improve the viability of chondrocytes by regulating the activity of downstream target molecules such as mTOR in articular chondrocytes.AMPK activity declining leads to the degeneration of articular cartilage and OA,and AMPK agonists can delay the development of experimental OA.All above studies report that the AMPK/mTOR signaling pathway in chondrocytes is one of the targets for the treatment of OA.In addition,autophagy in neuronal cells is likewise a therapeutic target for NP.Up-regulation autophagy in spinal dorsal root ganglion(DRG)can effectively alleviate NP brought about by peripheral nerve injury.The autophagy activator,rapamycin(mTOR inhibitor)exerts long-acting analgesic effect by increasing DRG autophagy in a dose-dependent manner to alleviate NP induced by spinal nerve ligation(SNL)in rats.Therefore,the occurrence and development of NP is closely concerned in autophagy of neuronal cells.However,whether the analgesic mechanism of medical ozone treatment of OA related to autophagy is not clear.Based on the previous study,the effects of intra-articular injection of different concentrations of medical ozone on OA in rats showed that ozone at 25 ?g/mL and 50?g/mL could increase the mechanical pain threshold of OA rats.Ozone of 25 ?g/mL could improve the smoothness of the cartilage surface,promote the number of chondrocytes and decrease the expression of matrix metalloproteinase-13(MMP-13).In vitro studies have found that low concentrations of medical ozone(? 30 ?g/mL)inhibited TNF-?,matrix metalloproteinase-3(MMP-3)and malondialdehyde(MDA)secretion of OA chondrocytes.All of the above demonstrate that low concentration of medical ozone has the function of anti-inflammatory,inhibition of cartilage degeneration and analgesia for OA.Therefore,our study hypothesized that the therapeutic effect of low concentration of medical ozone on OA regulates autophagy of chondrocytes through AMPK/mTOR signaling pathway,and inhibits the release of inflammatory factors and delays the degeneration of articular cartilage.And medical ozone activates AMPK in the spinal cord to exert the analgesic effect.Part I The effect of medical ozone on autophagy of cartilage in osteoarthritis rats and analgesic mechanismObjectiveIn this study,Monoiodoacetic Acid(MIA)-induced rat OA model was established and observed the effect of medical ozone on autophagy of cartilage and cartilage degeneration of OA.Neuropathic pain signaling pathways in spinal cord of OA were observed to explore the possible therapeutic mechanism and analgesic mechanism of medical ozone on OA.Methods 1.Experimental grouping and establishing an OA model.Female Wistar rats were randomly divided into 3 groups.Sham surgery group:50 ?L saline solution was injected into the right knee joint;MIA group:50 ?L of MIA was injected into the right knee joint to establish the OA model;MIA + O3 group:0.5 mL ozone(30 ?g/mL)was injected into the right knee joint on the 7th day,the 14th day and the 21st day of injection MIA.2.Detection of the general behavior,the mechanical withdrawal threshold(MWT)and the thermal withdrawal latency(PWL)of the rats.The general behavior,MWT(up-down method,Von Frey Hairs test)and PWL were observed and measured in each group on 1 day before MIA injection,and 1,4,7,14,21,28 days after injection.To observe the effect of medical ozone on pain behavior of OA rats.3.HE staining and Safranine O-fast green staining.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate.And then the right knee joint was taken down,fixed,decalcified,paraffin embedded,sliced and so on.HE staining and Safranine O-fast green staining were performed,and then Mankin score was measured to observe the effect of medical ozone on the degeneration of OA cartilage,4.The expression of cartilage MMP-13,collagen 2,LC3II and p62 were detected by immunohistochemistry.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate.And then the right knee joint was taken down,fixed,decalcified,paraffin embedded and sliced.Then,after dewaxing,hydration,antigen retrieval,blocking,primary and secondary antibody incubation,DAB color development,hematoxylin counterstaining and sealing,the expressions of MMP-13,collagen 2,LC3II and p62 were observed.To explore the effect of medical ozone on cartilage degeneration and cartilage autophagy.5.The expression of p-AMPK in the joint cartilage and spinal cord was detected by immunohistochemistry.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate,and then the right knee joint and L2-L4 spinal cord were cut from rats.After fixation,decalcification,paraffin embedding,sectioning,and then baked,dewaxed,hydrated,antigen repair,blocking,immune response,DAB color development,hematoxylin counterstaining,and sealing,the expression of p-AMPK in the cartilage and spinal cord was observed.To explore the possible therapeutic mechanism and analgesic mechanism of medical ozone on OA.6.The concentration of TNF-? and IL-6 in peripheral blood of rats was determined by EILSA.28 days after MIA injection,5 mL of peripheral blood was collected after anesthesia,and centrifuged at 3000 rpm for 10 min to obtain serum for testing.The standard samples,experimental samples and the antibody were added to the well of the test plate,respectively.And then the TMB color agent and the terminating agent were successively added after washing 3 times.In the end,the concentration of TNF-a and IL-6 were detected by a microplate reader.To explore the effect of medical ozone on the inflammation of OA.ResultsAt the 14 d,21 d and 28 d after MIA injection,the MWT and PWL in MIA group were significantly lower than those in the Sham group(P<0.01),and the MWT and PWL in the MIA+O3 group were significantly increased compared with the MIA group(P<0.01).The results of HE staining and Safranine O-Fast Green cartilage staining confirmed that injection of medical ozone delayed cartilage degeneration in OA.The Mankin score in the MIA group was significantly higher than that in the Sham group(P<0.01),and the Mankin score in the MIA+O3 group was significantly lower than that in the MIA group(P<0.01).Compared with MIA group,the expression of MMP-13 in cartilage was decreased,while the expression of collagen 2 was increased in the MIA+O3 group(P<0.05).The expression of LC3? in the MIA+O3 group was significantly higher than that in the MIA group(P<0.01),while the expression of p62 was lower than that in the MIA group(P<0.05).Compared with the Sham group,the expression of p-AMPK in cartilage was significantly decreased in the MIA group(P<0.01),and was significantly higher in the MIA+O3 group than that in the MIA group(P<0.01).Compared with the Sham group,the expression of p-AMPK in the spinal cord decreased in the MIA group(P<0.05),and significantly increased in the MIA+O3 group(P<0.05).Compared with the MIA group,the expression of p-AMPK of spinal cord in the MIA+O3 group was significantly increased(P<0.01).Compared with the Sham group,the concentration of IL-6 in the MIA group was significantly increased(P<0.01),and the concentration of TNF-? was increased(P<0.05).Compared with the MIA group,the concentration of IL-6 in the MIA+O3 group was decreased(P<0.01),and the concentration of TNF-? was decreased(P<0.05).The results showed that medical ozone had a certain inhibitory effect on OA inflammation.ConclusionInjection of medical ozone can effectively alleviate pain of OA,reduce the concentration of inflammatory factors IL-6 and TNF-?.inhibit the expression of MMP-13 and the degradation of collagen 2,up-regulate autophagy-associated protein LC3II and down-regulate p62 and delay cartilage degeneration in OA.These effects may be related to activation AMPK of spinal in addition to upregulation of autophagy in OA chondrocytes.Part ? Ozone induces Autophagy in Rat Chondrocytes Stimulated with IL-1? through the AMPK/mTOR Signaling PathwayObjectiveIn this study,primary rat chondrocytes were induced by IL-1?,and then treated with medical ozone to observe the effect of medical ozone on IL-1?-induced autophagy and the function of chondrocytes.To explore the mechanism of ozone on IL-1?-induced chondrcytes.Method1.Extraction,culture and identification of primary rat chondrocytes.2.CCK-8 method was used to detect the effect of different concentrations of medical ozone on cell viability of IL-1?-induced chondrocytes.The effect of autophagy inhibitor 3-methyladenine(3MA)and AMPK inhibitor compound C(com C)on survival ability of IL-1?-induced chondrocytes was detected by CCK-8.3.Western blotting and immunofluorescence were used to detect the effect of medical ozone on autophagy marker LC3II and p62 in IL-1?-induced chondrocytes.4.Quantitative Real-Time PCR(qRT-PCR)was used to detect the mRNA expression of TNF-a and IL-6,MMP-13 and TIMP-1.To observe the effects of medical ozone on the inflammation and degeneration of IL-1?-induced chondrocytes.5.Western blotting was used to detect AMPK/mTOR signaling pathway,autophagy-related proteins Beclin-1,ULK-1 and apoptosis-related proteins Bcl2 and Bax.To investigate whether medical ozone induces autophagy in IL-1?-induced chondrocytes by the AMPK/mTOR signaling pathway.6.Transmission electron microscopy(TEM)was used to observe the effect of medical ozone on autophagosomes in IL-1?-induced chondrocytes.Results1.The growth and identification of rat primary chondrocytes.Under inverted phase contrast microscope,the cells were observed to be triangular,quadrangular and polygonal.The cells were cultured for 2-3 days,and the cells were fused to about 70-80%,which showed a cobblestone-like appearance.The cell purity detected by cellular immunofluorescence is more than 95%.2.30 ?g/mL medical ozone increases the cell viability of chondrocytes induced by IL-1?.The cell viability of chondrocytes induced by IL-1? treatment with ozone was detected by CCK-8.The results showed that 40-60 ?g/mL of medical ozone inhibited the cell ability of chondrocytes induced by IL-1?,and the degree of inhibition was ozone concentration-dependent.Compared with the IL-1? group,30 ?g/mL of medical ozone promoted cell viability of chondrocytes(P<0.01),while the 50 ?g/mLg and 60 ?g/mL group of medical ozone inhibited cell viability of chondrocytes(P<0.05).To investigate whether medical ozone enhances cell viability of IL-1?-induced chondrocytes related to autophagy,chondrocytes with IL-1? were pretreated with 3MA,an autophagy inhibitor,before ozone treatment.Compared with the Control group,the cell ability of IL-1? group,IL-1?+3MA+o3 group and IL-1?+3MA group were significantly lower(P<0.01).There was no statistical difference between the IL-1?+O3 group and the Control group.The IL-1?+O3 group.the IL-1?+3MA+O3 group and IL-1?+3MA group had no significant difference compared with the IL-1? group.To investigate whether medical ozone enhances cell viability of IL-1?-induced chondrocytes associated with activation of AMPK,the chondrocytes induced by IL-1? were pretreated with com C,an AMPK inhibitor,before ozone treatment.Compared with the Control group,the IL-1? group,theIL-1?+com C+O3 group and the IL-1?+com C group were significantly lower;compared with the IL-1? group,the IL-1?+O3 group significantly increased(P<0.01).There was no significant difference in cell viability of the IL-1?+com C+O3 group and the IL-1?+com C group compared with the IL-1? group.3,30 ?g/mL medical ozone significantly increases autophagy in IL-1?-induced chondrocytes.Western blotting showed that compared with the Control group,LC3II was significantly decreased in the IL-1? group,IL-1?+3MA+O3 group and IL-1?+3MA group(P<0.01),and LC3II of the IL-1?+O3 group was also decreased(P<0.05).LC3? was increased in the IL-1?+O3 group compared with the IL-1? group(P<0.05).The expression of p62 in IL-1?+O3 group was significantly decreased(P<0.01)compared with IL-1? group.The 3MA pretreatment group inhibited the increase of LC3? and decreased p62 by medical ozone.The results of immunofluorescence showed that 30 ?g/mL medical ozone increased LC3? of IL-1?-induced expression in chondrocytes and inhibited p62,suggesting that autophagy of chondrocytes was promoted by medical ozone.3MA pretreatment inhibited the effect of ozone on autophagy in IL-1?-induced chondrocytes.4.30 ?g/mL medical ozone enhances autophagy of IL-1?-stimulated chondrocytes by the AMPK/mTOR signaling pathwayWestern blotting results showed that compared with the Control group,the expression of p-AMPK was decreased and the expression of p-mTOR was increased significantly in the IL-1? group(P<0.01).The level of p-AMPK was significantly higher in the IL-1?+O3 group than that in the IL-1? group,and the p-mTOR level was significantly lower(P<0.01).The Beclin-1 was considerably increased under the intervention of medical ozone(P<0.01),and 3MA inhibited the up-regulation of Beclin-1 by medical ozone.5.30 ?g/mL medical ozone inhibits the mRNA expression of IL-6 and TNF-? in IL-1?-induced chondrocytes.The mRNA expression of IL-6 and TNF-? in chondrocytes was detected by qRT-PCR.The results showed that the mRNA expression of IL-6 in IL-1?+O3 group was lower than that in IL-1? group(P<0.05).The mRNA expression of IL-6 in IL-1?+3MA group maintained a higher level than the Control group and the IL-1?group.The mRNA expression of TNF-? in IL-1?+O3 group was significantly lower than that in IL-1? group(P<0.01),while the mRNA expression of TNF-? in IL-1?+3MA group was higher than that in IL-1? group,but the difference was not statistically significant.6.Compound C inhibits the up-regulation of IL-1?-induced autophagy in chondrocytes and inhibits apoptosis.Western blotting and immunofluorescence showed that LC3? decreased and p62 increased in IL-1?+com C+O3 group compared with the Control group.In contrast,LC3? was significantly increased and p62 was decreased in the IL-1?+O3 group compared with that in the IL-1? group.Western blotting results showed that compared with the Control group,the expression of p-AMPK was significantly reduced in IL-1?group(P<0.01),and the expression of p-AMPK still kept the low level with com C pretreatment.Compared with the IL-1? group,the level of p-AMPK in the IL-1?+O3 group and the IL-1?+com C+O3 group were significantly increased(P<0.01),but the level of p-AMPK in IL-1?+O3 group was higher than that in IL-1?+com C+O3 group,suggesting that com C partially inhibited the up-regulation of p-AMPK by medical ozone.The p-mTOR in the IL-1? group and IL-1?+com C+O3 group were significantly higher than those in the Control group(P<0.01),and p-mTOR in the IL-1?+O3 group was significantly lower than that in the IL-1? group(P<0.01).the expression of ULK-1 was higher in the IL-11?+O3 group than that in the IL-1? group(P<0.05).Compared with IL-1? group,Bcl2/Bax was markedly increased in IL-1?+O3 group(P<0.01).7.Compound C inhibits medical ozone down-regulation the mRNA expression of IL-6 and TNF-? in IL-1?-induced chondrocytes.The expression of IL-6 and TNF-? mRNA in chondrocytes was detected by qRT-PCR.The results showed that IL-6 and TNF-? mRNA expression were up-regulated in IL-1? group compared with the Control group.Compared with IL-1?group,the mRNA expression of IL-6 and TNF-? in IL-1?+O3 group were significantly down-regulated(P<0.01).The mRNA expression of IL-6 and TNF-? in IL-1?+com C group increased compared with IL-1? group.The mRNA expression of IL-6 in IL-1?+com C+O3 group was higher than that in IL-1? group,but the mRNA expression of TNF-a was significantly lower than that in IL-1? group(P<0.01).8.Compound C inhibitis down-regulation the mRNA expression of MMP-13 and up-regulation the mRNA expression of TIMP-1 in IL-11?-induced chondrocytes by medical ozone.The results of qRT-PCR showed that compared with IL-1? group,the mRNA expression of MMP-13 in IL-1?+O3 group was significantly decreased(P<0.01),and the mRNA expression of TIMP-1 was significantly increased(P<0.01).The mRNA expression of MMP-13 in IL-1?+com C+O3 group was significantly decreased(P<0.01),and the expression of TIMP-1 mRNA was increased(P<0.05),indicating that com C inhibited the regulating the expression of MMP-13 and TIMP-1 in chondrocytes by medical ozone.9.30 ?g/mL medical ozone promotes the formation of autophagosomes in chondrocytes induced by IL-1?.Transmission electron microscopy showed that the number of autophagosomes in the IL-1? group was decreased,and the number of autophagosomes in the IL-1?+O3 group was significantly increased.The number of autophagosomes in IL-?+3MA+O3 group and IL-?+com C+O3 group was not significantly different from that of the IL-1? group.ConclusionMedical ozone enhances autophagy of IL-1?-induced chondrocytes by activating the AMPK/mTOR signaling pathway.In addition,medical ozone inhibits the transcription of inflammatory factors in IL-1? induced chondrocytes,inhibits the decomposition of extracellular matrix and promotes synthesis of chondrocytes,which provides an experimental basis for the effective treatment of OA in medical ozone. |
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