Objective:To discuss the regulatory effect of lutein on the phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)signaling pathway and chondrocyte autophagy in mandibular joint cartilage tissue of rabbits with traumatic temporomandibular joint ankylosis(TMJA),and to clarify its mechanism.Methods:Thirty-two male New Zealand white rabbits were divided into sham operation group,model group,lutein group,and 3-MA(PI3K/AKT signaling pathway inhibitor)+lutein group,and there were 8 rabbits in each group.The rabbit model of TMJA was established in model group,lutein group,and 3-MA+lutein group,while the rabbits in sham operation group only underwent tissue exposure without surgery.The rabbits in lutein group were administered 10 mg·kg-1 lutein,and those in 3-MA+lutein group were administered 15 mg·kg-1 3-MA and 10 mg·kg?1 lutein.All the drugs were injected via the marginal ear vein starting 24 h after surgery,once a week for 3 consecutive months.After completion,the cartilage tissue on the surgical side was collected.HE staining was used to evaluate the patho morphology of the mandibular joint cartilage tissue of the rabbits in various groups;Western blotting method was used to detect the expression levels of PI3K,AKT,phosphorylated AKT(p-AKT),Beclin-1,autophagy-related protein 5(ATG5),microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ),microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),autophagy receptor protein(P62),matrix metalloproteinase 13(MMP-13),a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5),aggrecan,and type Ⅱ collagen(Col Ⅱ)proteins in temporomandibular joint cartilage tissue of the rabbits in various groups;transmission electron microscopy was used to detect the numbers of autophagosomes in temporomandibular joint cartilage tissue of the rabbits in various groups.Results:Compared with sham operation group,the pathological score of mandibular joint cartilage tissue of the rabbits in model group was increased(P<0.05).The Western blotting results showed that compared with sham operation group,the expression levels of PI3K,p-AKT,aggrecan,and Col Ⅱ proteins in mandibular joint cartilage tissue of the rabbits in model group were decreased(P<0.05),while the expression levels of Beclin-1,ATG5,P62,MMP-13,ADAMTS-5 proteins and ratio of LC3-Ⅱ/LC3-Ⅰ were increased(P<0.05).Compared with model group,the expression levels of PI3K,p-AKT,aggrecan,and Col Ⅱ proteins in mandibular joint cartilage tissue of the rabbits in lutein group were increased(P<0.05),while the expression levels of Beclin-1,ATG5,P62 protein and ratio of LC3-Ⅱ/LC3-Ⅰ were decreased(P<0.05).Compared with lutein group,the expression levels of PI3K,p-AKT,Beclin-1,ATG5,and ratio of LC3-Ⅱ/LC3-Ⅰ,and P62 proteins in mandibular joint cartilage tissue of the rabbits in 3-MA+lutein group were decreased(P<0.05),while the expression levels of MMP-13 and ADAMTS-5 proteins were increased(P<0.05),and the expression levels of aggrecan and Col Ⅱ proteins were decreased(P<0.05).The transmission electron microscope observation results showed that compared with sham operation group,the number of autophagosomes in temporomandibular joint cartilage tissue of the rabbits in model group was increased(P<0.05);compared with model group,the number of autophagosomes in temporomandibular joint cartilage tissue of the rabbits in lutein group was decreased(P<0.05);compared with lutein group,the number of autophagosomes in temporomandibular joint cartilage tissue of the rabbits in 3-MA+lutein group was decreased(P<0.05).Conclusion:Lutein improves the mandibular joint cartilage tissue damage in the TMJA rabbits by regulating the PI3K/AKT signaling pathway and chondrocyte autophagy.

BACKGROUND:Due to its unknown etiology and the lack of definitive curative treatments,management of temporomandibular joint osteoarthritis primarily focuses on symptom relief.Therefore,the identification of effective early molecular diagnostic biomarkers or potential therapeutic targets holds great significance.OBJECTIVE:To investigate the expression of mitochondrial creatine kinase 2 in temporomandibular joint osteoarthritis in rats and its role in the progression of inflammation.METHODS:(1)Animal experiment:Twenty Sprague-Dawley rats were randomly divided into control and unilateral anterior crossbite groups(n=10).A rat model of temporomandibular joint osteoarthritis was made in the unilateral anterior crossbite group.Four weeks after modeling,histological evaluations,including hematoxylin-eosin and Safranin O-fast green staining,were performed to assess pathological changes in the cartilage and subchondral bone of the mandibular condyle.Quantitative real-time PCR and immunohistochemical staining were utilized to detect the mRNA and protein expression levels of interleukin-1β,matrix metalloproteinase 13,type II collagen,aggrecan,and mitochondrial creatine kinase 2 in condylar cartilage.(2)Cell experiment:Passage 3 condylar cartilage cells from Sprague-Dawley rats were divided into control group and model group.Cells in the control group were routinely cultured,while an inflammation model of condylar cartilage cells was established with interleukin 1β in the model group were treated with interleukin-1β to induce inflammatory cell models.After 24 hours of interleukin-1β treatment,western blot was used to evaluate the expression of matrix metalloproteinase 13,type II collagen proteins in chondrocytes.Western blot was also used to detect the protein expression of mitochondrial creatine kinase 2 in the model group at 0,12,24,48,and 72 hours after treatment with interleukin-1β.RESULTS AND CONCLUSION:(1)Animal experiment:The results of hematoxylin-eosin staining showed that the unilateral anterior crossbite group exhibited disintegration of the fibrous layer in the cartilage of the mandibular condyle,disorganization of chondrocyte hierarchy,dense cellularity of the proliferative layer,obvious cell clustering,and infiltration of inflammatory cells in the subchondral bone.The results of Safranin O-fast green staining showed that in the cartilage matrix in the unilateral anterior crossbite group,the red stain became less extensive and lighter in color,and the tidal line was discontinuous.The mRNA and protein expression levels of interleukin-1β and matrix metalloproteinase 13 were elevated in the unilateral anterior crossbite group compared with the control group(P<0.05).Conversely,the relative expression of type II collagen,aggrecan,and mitochondrial creatine kinase 2 mRNA and protein decreased(P<0.05).(2)Cell experiment:Compared with the control group,the protein expression of matrix metalloproteinase 13 in the experimental group was significantly increased(P<0.05),while the protein expression of type II collagen was decreased(P<0.05).With prolonged interleukin-1β treatment,the protein expression of mitochondrial creatine kinase 2 in condylar chondrocytes of the model group gradually decreased.All the results indicate that the relative expression level of mitochondrial creatine kinase 2 was negatively correlated with the degree of chondrocyte inflammation in the rat model of temporomandibular joint osteoarthritis.Therefore,it is reasonable to infer that mitochondrial creatine kinase 2 is an important indicator for assessing the progression of inflammation in temporomandibular joint osteoarthritis.

BACKGROUND:Due to its unknown etiology and the lack of definitive curative treatments,management of temporomandibular joint osteoarthritis primarily focuses on symptom relief.Therefore,the identification of effective early molecular diagnostic biomarkers or potential therapeutic targets holds great significance.OBJECTIVE:To investigate the expression of mitochondrial creatine kinase 2 in temporomandibular joint osteoarthritis in rats and its role in the progression of inflammation.METHODS:(1)Animal experiment:Twenty Sprague-Dawley rats were randomly divided into control and unilateral anterior crossbite groups(n=10).A rat model of temporomandibular joint osteoarthritis was made in the unilateral anterior crossbite group.Four weeks after modeling,histological evaluations,including hematoxylin-eosin and Safranin O-fast green staining,were performed to assess pathological changes in the cartilage and subchondral bone of the mandibular condyle.Quantitative real-time PCR and immunohistochemical staining were utilized to detect the mRNA and protein expression levels of interleukin-1β,matrix metalloproteinase 13,type II collagen,aggrecan,and mitochondrial creatine kinase 2 in condylar cartilage.(2)Cell experiment:Passage 3 condylar cartilage cells from Sprague-Dawley rats were divided into control group and model group.Cells in the control group were routinely cultured,while an inflammation model of condylar cartilage cells was established with interleukin 1β in the model group were treated with interleukin-1β to induce inflammatory cell models.After 24 hours of interleukin-1β treatment,western blot was used to evaluate the expression of matrix metalloproteinase 13,type II collagen proteins in chondrocytes.Western blot was also used to detect the protein expression of mitochondrial creatine kinase 2 in the model group at 0,12,24,48,and 72 hours after treatment with interleukin-1β.RESULTS AND CONCLUSION:(1)Animal experiment:The results of hematoxylin-eosin staining showed that the unilateral anterior crossbite group exhibited disintegration of the fibrous layer in the cartilage of the mandibular condyle,disorganization of chondrocyte hierarchy,dense cellularity of the proliferative layer,obvious cell clustering,and infiltration of inflammatory cells in the subchondral bone.The results of Safranin O-fast green staining showed that in the cartilage matrix in the unilateral anterior crossbite group,the red stain became less extensive and lighter in color,and the tidal line was discontinuous.The mRNA and protein expression levels of interleukin-1β and matrix metalloproteinase 13 were elevated in the unilateral anterior crossbite group compared with the control group(P<0.05).Conversely,the relative expression of type II collagen,aggrecan,and mitochondrial creatine kinase 2 mRNA and protein decreased(P<0.05).(2)Cell experiment:Compared with the control group,the protein expression of matrix metalloproteinase 13 in the experimental group was significantly increased(P<0.05),while the protein expression of type II collagen was decreased(P<0.05).With prolonged interleukin-1β treatment,the protein expression of mitochondrial creatine kinase 2 in condylar chondrocytes of the model group gradually decreased.All the results indicate that the relative expression level of mitochondrial creatine kinase 2 was negatively correlated with the degree of chondrocyte inflammation in the rat model of temporomandibular joint osteoarthritis.Therefore,it is reasonable to infer that mitochondrial creatine kinase 2 is an important indicator for assessing the progression of inflammation in temporomandibular joint osteoarthritis.

Objective To investigate the effect of medical ozone injection therapy on temporomandibular joint(TMJ)osteoarthritis and its pain in SD rats.Methods Fifity-four rats were randomly assigned according to a random number table into three groups:con-trol group,model group,and medical ozone group,with 18 rats in each group.In the control group,only physiological saline was in-jected during modeling;in the model group,only sodium iodoacetate was injected for modeling;in the medical ozone group,after in-jecting sodium iodoacetate into the joint cavity for modeling for one week,medical ozone was then injected into the joint cavity for inter-vention at a frequency of once a week,totaling 5 times.One week(week 2 after modeling),3 weeks(week 4 after modeling),and 5 weeks(week 6 after modeling)after medical ozone injections,6 rats from each group were euthanized.Mechanical withdrawal thresh-old of rats in each group was assessed before euthanasia,and the expression levels of interleukin-1β(IL-1β)in joint fluid of rats in each group were measured after euthanasia.Gross observation and modified Mankin's scoring were performed on TMJ cartilage of rats in each group after stained with Pelletier score and Safranin O-Fast Green.Results During the same time period,compared to the control group,the model group showed a significant decrease in the mechanical pain threshold of the TMJ in rats at 1 week,3 weeks,and 5 weeks(P＜0.01).The expression levels of IL-1β in the TMJ fluid increased(P＜0.01),and the Pelletier score and modified Mankin's score of TMJ cartilage increased(P＜0.01).In comparison to the model group,the medical ozone group exhibited a significant increase in the mechanical pain threshold of the TMJ in rats after 3 weeks and 5 weeks of medical ozone injections(P＜0.01).The expression levels of IL-1β in the TMJ fluid decreased(P＜0.01),and the Pelletier score and modified Mankin's score of TMJ cartilage decreased(P＜0.01).However,there were no statistically significant difference in the measured parameters in the TMJ cavity after 1 week of medical ozone injection(P＞0.05).Within the medical ozone group,compared to the 1-week treatment,the mechanical pain threshold of the TMJ increased(P＜0.01)and the expression levels of IL-1β in the TMJ fluid decreased at 3 weeks and 5 weeks(P＜0.01).However,there was no statistically significant difference in the Pelletier score and modified Mankin's score of TMJ cartilage(P＞0.05)between different treatment duration.Additionally,there were no statistically signif-icant differences in the mechanical pain threshold of the TMJ,expression levels of IL-1β in the TMJ fluid,Pelletier score,and modi-fied Mankin's score of TMJ cartilage between the medical ozone group at 3 weeks and 5 weeks(P＞0.05).Conclusion Medical ozone treatment for more than 3 weeks can improve temporomandibular joint osteoarthritis and its associated pain in rats.