Study On Inflammatory Mechanism Of Preventing And Treating Cartilage Degeneration In Posttraumatic Osteoarthritis Secondary To Rabbit Acetabular Fractures By Icariin

BackgroundIn recent years,a characteristic health security service model of Traditional Chinese Medicine(TCM)with the core concept of"Prevention of disease"gradually taking shape.As a classical theory of TCM,“Prevention of disease”can be vigorously promoted and applied with the rapid development of modern science and technology,which fully shows the great charm and practicability of the classical theory of TCM.Posttraumatic osteoarthritis(PTOA)is one of the most common complications after acetabular fracture,which has a great impact on the postoperative function and quality of life of patients.The progress of PTOA disease is slow,so it is suitable for early preventing and treating by means of TCM under the guidance of the theory of“Prevention of disease”in TCM.Icariin(ICA)is the extract of common TCM Herba Epimedii.Previous studies have shown that ICA has a preventive and therapeutic effect on PTOA,but the specific mechanism is not clear.In this study,a rabbit model of secondary PTOA of acetabular fracture was established,and modern experimental techniques were used to explore the mechanism of ICA in the preventing and treating of secondary PTOA of acetabular fracture under the guidance of the theory of treatment of acetabular fracture in TCM,so as to provide theoretical and experimental basis for the clinical preventing and treating of PTOA after acetabular fracture in the future.Chapter 1 Study on prevalence of posttraumatic osteoarthritis after acetabular fracture fixationObjective:To explore the prevalence of posttraumatic osteoarthritis after acetabular fracture fixation through clinical retrospective study.Methods:The patients with acetabular fractures who underwent surgery in Central Theater General Hospital from January 1,2008 to December 31,2018 were investigated by EMRS system.Kellgren-Lawrence osteoarthritis standard(K-L standard)and ACR hip osteoarthritis standard(ACR standard)were used to evaluate PTOA,and the incidence of secondary PTOA after acetabular fracture operation was calculated.Results:A total of 381 patients with acetabular fractures were treated by Central Theater General Hospital from January 1,2008to December 31,2018.According to K-L standard and ACR standard,the incidence of postoperative PTOA of acetabular fracture was22.22%and 26.86%,respectively.Conclusion:The improvement of surgical technology of acetabular fracture does not significantly reduce the prevalence of posttraumatic osteoarthritis(PTOA.Therefore,there is an urgent need to improve the understanding of this problem and study the effective"Prevention of disease"method and its mechanism.Chapter 2 Establishment and verification of posttraumatic osteoarthritic model following fracture of acetabulum in rabbit Objective:To establish a rabbit model of secondary PTOA of acetabular fracture and verify it,so as to provide a research model for further discussion on the mechanism of preventing and treating of secondary PTOA of acetabular fracture by means of TCM under the guidance of the theory of"Prevention of disease"of TCM.Methods:Twelve adult male New Zealand rabbits were randomly divided into Sham group(n=4),Model group(n=4)and open reduction and internal fixation group(ORIF group,n=4).Except for Sham group,the posterior wall of acetabulum fractures were caused by operation in other rabbits.The ORIF group was treated with open reduction and internal fixation after acetabular fracture,while the Model group was not treated with reduction and fixation after acetabular fracture.All the experimental rabbits were killed 6m after modeling.The gross,X-ray and histopathological manifestations of the left hip joint of each group were qualitatively analyzed,and the X-ray and histopathological manifestations were quantitatively analyzed by OA radiological semi-quantitative score and Mankin histological score.Results:Of the 8 acetabular fracture models,7(87.5%)were simple posterior wall fractures,and the remaining one was combined fracture(posterior wall and transverse fracture).All rabbits had stable hip joints after acetabular fractures,and there was no sciatic nerve injury.The rabbits in Sham group and ORIF group returned to normal gait within 2 weeks after modeling.However,the rabbits in the Model group still showed obvious lameness and limited movement at 2 weeks after modeling.The left hip joint of the rabbits in the 6m model group showed obvious PTOA manifestations in gross view,X-ray and histopathology,compared with the ORIF group,the degree of PTOA was alleviated to a certain extent.There was significant difference in the semi-quantitative score of OA radiology among the three groups(P<0.05).Compared with Sham group,the scores of Model group and ORIF group were significantly higher(P<0.05),and the scores of ORIF group were not significantly lower than those of Model group(P>0.05).There was significant difference in Mankin histological score among the three groups(P<0.05).The pairwise comparison showed that Model group>ORIF group>Sham group,the difference was statistically significant(P<0.05).Conclusion:A rabbit acetabular fracture secondary PTOA model established in this study can show typical PTOA manifestations from three aspects of gross,X-ray and histopathology 6m after modeling,and has high consistency and repeatability,which provides a new and reliable animal model for the study of the mechanism of preventing and treating of PTOA by using the theories and methods of TCM.Chapter 3 Study on inflammatory mechanism of preventing and treating cartilage degeneration in posttraumatic osteoarthritis secondary to rabbit acetabular fractures by IcariinObjective:On the basis of the model established in chapter2,through animal experiments in vivo,to explore the inflammatory mechanism of ICA in the preventing and treating of cartilage degeneration secondary to acetabular fracture in rabbits under the guidance of TCM treatment theory.Methods:60 adult male New Zealand rabbits were randomly divided into Control group,Sham group,Model group,ORIF group and ICA group,with 12 rabbits in each group.Except for Control group and Sham group,the posterior wall of acetabulum fractures were caused by operation in other rabbits.ORIF group and ICA group were treated with open reduction and internal fixation after acetabular fracture,Model group was not fixed after acetabular fracture,ICA group was given ICA(60mg/kg/day)after modeling,and the other groups were given the same amount of normal saline.The experimental rabbits were randomly killed at 3 days,3 weeks and 6 m after modeling,and4 experimental rabbits were killed in each group at each time point.The levels of inflammatory cytokines IL-1?and TNF-?in synovial fluid and synovium of left hip joint were detected by enzyme linked immunosorbent assay(ELISA),and the protein and m RNA expression levels of key molecules of NF-?B signal pathway in synovial tissue of left hip joint were detected by Western blot and real time fluorescence quantitative polymerase chain reaction(RT-PCR),respectively.Western blot and RT-PCR were used to detect the protein and m RNA expression of catabolic metabolic markers MMP-1,MMP-13 and synthetic metabolic markers Aggrecan and collagen?,in situ terminal transferase labeling(Tunel)was used to detect the level of chondrocyte apoptosis,and hematoxylin-eosin(He)staining and toluidine blue staining were used to detect the morphological changes of articular cartilage.Results:The comparison of inflammatory cytokines(IL-1?,TNF-?),inflammatory pathway(NF-?B signal pathway),catabolic metabolic markers(MMP-1,MMP-13),anabolic metabolic markers(Aggrecan,Collagen?)and apoptosis index of chondrocytes showed that there were significant differences in the above indexes among the groups at 3 days,3 weeks and 6 m after modeling(P<0.05).The results of pairwise comparison showed that there was no significant difference in the above-mentioned indexes between the Sham group and the Control group at 3 days,3 weeks and 6 m after the establishment of the model(P>0.05).However,the expression of other indexes except synthetic metabolic markers in Control group,Model group,ORIF group and ICA group were all in the order of Model group>ORIF group>ICA group>Control group.The results of pairwise comparison of synthetic metabolic markers were opposite,and the differences were statistically significant(P<0.05).The comparison of inflammatory cytokines,NF-?B signal pathway,catabolic markers,anabolic markers and chondrocyte apoptosis index showed that there was no significant difference between Control group and Sham group at 3 time points(P>0.05),but there was significant difference among Model group,ORIF group and ICA group(P<0.05).The results of pairwise comparison showed that the content of inflammatory factors increased first and then decreased with time(3d<6m<3w).NF-?B pathway,catabolic markers and apoptosis index of chondrocytes gradually increased with time(3d<3w<6m),while the expression of anabolic markers decreased gradually with time(3d>3w>6m).The differences were statistically significant(P<0.05).The results of histomorphology of hip cartilage in each group showed that there was no significant degeneration between Control group and Sham group at 3 time points.However,the morphological degeneration of articular cartilage in Model group,ORIF group and ICA group was aggravated gradually with time.In Model group,obvious signs of injury and rupture of articular surface were observed at 3 days after modeling,but there were no obvious abnormalities in the number,morphology,distribution and arrangement of chondrocytes.At 3 weeks after modeling,there were still signs of injury and rupture of articular surface,rough surface of articular surface,thinning of cartilage layer and slight decrease in the number of chondrocytes.The distribution was slightly uneven and the arrangement was slightly disordered,but at6m,the articular cartilage had no obvious articular surface structure,huge cystic changes in cartilage tissue,obvious abnormal morphology of chondrocytes,and few chondrocytes in normal shape.a large number of fibroblasts proliferated,the distribution of cells was seriously uneven,the arrangement was very disordered,and there was obvious articular cartilage degeneration.At 3 days and 3 weeks after modeling,there was no significant difference in cartilage degeneration among ORIF group,ICA group and Model group,but at 6 m after modeling,ICA group was better than ORIF group and Model group.Conclusion:The results of animal studies in vivo show that the abnormal expression of joint inflammatory factors and inflammatory pathways may be one of the important mechanisms leading to PTOA cartilage degeneration secondary to acetabular fractures in rabbits.Under the guidance of the theory of"Prevention of disease"in TCM,the early intervention of ICA may improve the metabolic imbalance and chondrocyte apoptosis of articular cartilage tissue by affecting the expression of inflammatory factors(IL-1?,TNF-?)and inflammatory pathway(NF-?B signal pathway)in experimental rabbits,and then reduce the cartilage degeneration and the degree of PTOA of the model secondary to acetabular fracture in rabbits.This shows that the theory of"Prevention of disease"in TCM is still scientific and practical.Chapter 4 Effect of Icariin on inflammatory phenotype of human chondrocytes induced by IL-1?in vitroObjective:To explore the intervention mechanism of ICA pretreatment on the inflammatory phenotype of human chondrocytes(HC-A)induced by IL-1?under the guidance of“Prevention of disease”of TCM in vitro.Methods:HC-A were routinely resuscitated,cultured and passaged.1?3 generations of HC-A were used in the experiment in vitro.The best concentration of ICA on HC-A was detected by CCK-8.Control group was treated with normal medium;Model group was treated with medium containing 1ng/ml IL-1?in HC-A for 24 hours,and ICA group was treated with medium containing 1ng/ml IL-1?+10^-9mol/LICA in HC-A for 24 hours.The effects of ICA on inflammatory mediators PGE-2,NO,NF-?B signal pathway,chondrocyte metabolic markers and chondrocyte apoptosis induced by IL-1?in HC-A were observed.Results:The results of CCK-8 detection showed that 10^-15?10^-9mol/LICA had no obvious toxic effect on HC-A and no significant effect on cell viability,and the cell viability was the best when10^-9mol/LICA acted on HC-A.After treatment with IL-1?,the expression levels of PGE-2,NO,P-NF?B p65,P-I?B?,IKK?,MMP-1,MMP-13 and apoptosis index of HC-A were significantly increased(P<0.05).Compared with HC-A treated with IL-1?alone,the expression levels of PGE-2,NO,P-NF?B p65,P-I?B?,IKK?,MMP-1,MMP-13 and HC-A apoptosis index of HC-A pretreated with 10^-9mol/LICA decreased significantly(P<0.05).The results of Aggrecan and Collagen?were on the contrary,and the differences were statistically significant(P<0.05).Conclusion:HC-A is induced by IL-1?to simulate the inflammatory reaction of PTOA and pretreated with ICA.It is verified that ICA preconditioning may inhibit the metabolic imbalance and apoptosis of HC-A by inhibiting the overexpression of inflammatory mediators(PGE-2,NO)and the activation of NF-?B signal pathway.It provides in vitro cell experimental evidence for the early intervention of ICA in the preventing and treating of secondary PTOA of acetabular fracture under the guidance of the"Prevention of disease"theory of TCM,and also provides a further theoretical and experimental basis for the clinical application of ICA in the preventing and treating of PTOA in the future.