| Chapter 1 The effect of different intensity treadmill exercise on the cartilage,subchondral bone and infrapatellar Fat Pad of knee joint in ratsObjective: To investigate the dynamic effects of treadmill running at different intensities on articular cartilage,subchondral bone and infrapatellar Fat Pad of rats,and to understand the effects of treadmill exercise with different intensities on knee joint health from two perspectives: time(different time points)and space(different joint components).Methods: A total of 72 adult SD rats were randomly divided into Sedentary group(SED),Low-intensity running group(LIR),Medium-intensity running group(MIR)and High-intensity running group(HIR).The animals of SED were housed in cages,while the running groups were carried out treadmill running according to their respective exercise plan.All animals were examined at 1week,4 weeks,and 8 weeks as follows: Staining methods including hematoxylin-eosin,Safranin O-Fast Green Staining,and Sirius red were used to detect histopathological changes of articular cartilage.Expression of genes related to cartilage synthesis and decomposition was detected by RT-PCR,and apoptosis of chondrocytes was observed by Caspase3 immunohistochemical staining.Micro-CT was used to evaluate the structural parameters of subchondral bone,TRAP and toluidine blue staining were used to evaluate the morphological changes and bone metabolism of subchondral bone,and RT-PCR was used to detect the expression of osteogenic and osteoclast-related genes.HE and Masson staining were used to evaluate the histomorphology changes and fibrosis of infrapatellar fat pad;RT-PCR was used to detect the expression of fat and inflammation related genes;CD26 and CD206 immunohistochemical staining were used to evaluate the composition of immune cells in infrapatellar fat pad.Results: 1.Effect on articular cartilage: After 1 week’s running training,the tibial articular cartilage of LIR group was the thickest,which was significantly different from that of HIR(P < 0.01)and SED groups(P < 0.001);the Makin’s score of tibial plateau articular cartilage of LIR and MIR groups was significantly lower than that of SED and HIR groups,The m RNA of ADAMTS5 in HIR group was significantly higher than that in SED,LIR and MIR groups.;After 4 week’s running training,the thickness of tibial articular cartilage in all running groups was higher than that in SED group,but there was no significant difference in other histological changes among all groups.The m RNA expression of MMP3 in HIR group was significantly higher than that in SED group(P<0.001)and LIR group(P<0.001).The expression of COL2α m RNA in MIR group was significantly higher than that in all running groups and SED group(P<0.001).After 8 week’s running training,the cartilage surface of the tibial plateau cartilage contact area in HIR group was irregular,the surface cartilage was missing,the collagen arrangement was uneven,and the pigmentation of the cartilage matrix was shallow and the tide line was blurred in the Safranin O-Fast Green Staining.The thickness of femoral cartilage in the MIR and LIR groups was thicker than that in the SED and HIR groups,and the difference was statistically significant.Makin’s score in LIR(2.81±1.96),MIR(1.60±0.74)and SED(2.70±0.97)groups were significantly lower than that in HIR(5.80±3.61)group.The IOD of apoptotic protein Caspase3 in HIR group(0.177±0.013)was significantly higher than that in MIR group(0.126±0.028,P=0.045).The m RNA expression of ADAMTS 5 in the MIR and the SED group was significantly higher than that in the LIR group(P<0.05).The m RNA expression of MMP3 in HIR group was significantly higher than that in SED group(P<0.01)and MIR group(P<0.01).Compared with SED group,the SOX9 m RNA expression was significantly lower in MIR group and HIR group(P<0.001).The m RNA expression levels of ACAN and COL2ɑ were significantly higher in the LIR group than in the other groups(P<0.001).2.Effect on subchondral bone: After 1 week’s running training,Micro CT showed that the medial BMD of femoral subchondral bone plate in HIR group(0.980±0.001)was significantly lower than that in SED group(1.084±0.001,P =0.001).Meanwhile,the thickness of medial femoral subchondral plate in HIR group(0.087±0.006)was thinner than that in MIR group(0.132±0.014)and SED group(0.131±0.014),and the porosity in HIR group(65.25±9.87)was higher than that in MIR(32.34±11.52)and SED group(33.70±5.37).The femoral subchondral cancellous bone trabecular septum(Tb.Sp)in HIR group was significantly lower than that in LIR group.Tb.Sp in the lateral part of subchondral cancellous bone of tibia in the MIR group was significantly lower than that in the HIR and SED groups,and BMD in the medial part of subchondral cancellous bone of tibia in the MIR group was significantly lower than that in the SED group.The CTSK m RNA and Runx2 m RNA of MIR were significantly higher than those of other intensity groups,the difference was statistically significant.The expression OPN m RNA in LIR group and HIR group was significantly lower than that in SED group.After 4 week’s running training,Micro-CT detection showed that the thickness of the medial femoral subchondral bone plate in the LIR group(0.117±0.009)was significantly lower than that in the SED group(0.154±0.019,P=0.029).BMD of femoral subchondral bone in MIR and HIR group was significantly lower than that in SED group,and Tb Th of femoral subchondral bone in MIR group was significantly lower than that in SED group.BMD of subchondral tibia in LIR group was significantly lower than that in SED group,BMD of subchondral tibia in MIR and HIR group was significantly lower than that in SED group,and Tb Th of subchondral tibia in LIR group was significantly lower than that in SED group.The expression of TRAP in LIR group was significantly higher than that in other intensity running groups and SED group,and the difference was statistically significant.After 4 week’s running training,Micro-CT showed that BMD(1.140±0.018)in LIR group was significantly lower than that in SED group(1.238±0.006,P=0.009).In the medial tibial subchondral bone plate,the porosity of HIR group(25.54±0.46)and LIR group(15.66±0.75)was significantly lower than that of MIR group(30.94±0.46)and SED group(32.06±0.43).BMD and Tb.Sp in LIR group were significantly lower than those in SED group,but connection density(CD)and Tb Th in LIR group were significantly higher than those in HIR group.BMD of lateral subchondral plate of tibia in MIR group was significantly lower than that in SED group.The expression of osteoclast related gene TRAP in LIR group was significantly higher than that in MIR group and SED group,and the expression of osteoclast related gene TRAP in HIR group was significantly higher than that in all intensity running group and SED group.The difference was statistically significant.3.Effect on infrapatellar fat pad: After 1 week’s running training,the expression of serum visfatin in LIR group(12.22±0.29ng/ml)and Mir group(10.90±1.09 ng/ml)was significantly higher than that in sed group(7.65±1.12 ng/ml)and HIR group(9.31±0.93ng/ml),respectively.The expression of visfatin in HIR group was significantly higher than that in SED group.The thickness and number of cells on the surface of infrapatellar fat pad(IFP)in HIR group(2.00 ± 1.41)were significantly greater than those in MIR group(0.33 ± 0.57).MIR group had the lowest degree of fibrosis,HIR group had the highest degree of fibrosis,and there was significant difference between the two groups.After 4 week’s running training,there was no significant difference in histological staining among the groups.RT-PCR showed that the m RNA expression of HOXC9 and PPARg in the MIR group was significantly higher than that in the SED group.After 8 week’s running training,the IFP surface cell infiltration score in LIR,MIR and SED groups was significantly lower than that in HIR group(P<0.05),and the adipocyte diameter in MIR group(0.0389±0.0004mm)was significantly higher than that in HIR group(0.0330±0.0012mm).Compared with the SED group(1.12±0.10),the MIR group(0.34±0.29,P=0.021)and LIR group(0.33±0.33,P=0.020)had significantly decreased fibrosis scores.In addition,we also compared the fibrosis scores of each intensity group at 8 weeks and the 1-week SED group,and the results showed that the HIR group(1.29±0.36)was significantly higher than the 1-week SED group(0.54±0.54;P = 0.019).IFP vessel density in HIR group(33.31±8.43)and SED group(27.58±5.36)was significantly higher than that in LIR group(16.00±2.00)and MIR group(15.00±6.44).Conclusion: 1.The effect of treadmill exercise on articular cartilage,subchondral bone and infrapatellar fat pad is time and intensity-dependent.2.High intensity treadmill exercise can lead to articular cartilage degeneration,abnormal subchondral bone remodeling,and increased fibrosis of infrapatellar fat pad.The change of subchondral bone was earlier than that of articular cartilage and infrapatellar fat pad.Chapter 2 The role of Wnt/β-catenin signaling pathway in the interaction of articular cartilage and subchondral bone during medium intensity runningObjective: To investigate the activation of Wnt/β-catenin signaling pathway in subchondral bone after different intensity treadmill running,and to further clarify the role of this signaling pathway in the interaction between articular cartilage and subchondral bone of knee joint in rats under medium intensity treadmill running.Methods: The activation of Wnt/β-catenin signaling pathway related proteins and m RNA in subchondral bone of knee joint in different intensity running group at different time points was detected by RT-PCR and Western Bloting.Then,the Wnt/β-catenin signaling pathway specific inhibitor DKK1 was intra-injected into the knee joint,in order to evaluate the changes of cartilage and subchondral bone in the medium running group after DKK1 injection to further verify the role of Wnt/β-catenin signaling pathway in the interaction of articular cartilage and subchondral bone under medium intensity treadmill running.Results: 1.8 weeks of medium-intensity running could activate the Wnt/β-catenin signaling pathway in subchondral bone.At the protein level,the expression of GSK3β protein in LIR group and MIR group was significantly lower than that in SED group(P < 0.05).At m RNA level,the expression of β-catenin in LIR group was significantly higher than that in SED group(P < 0.05),and the expression of β-catenin and LRP6 in MIR group were significantly higher than that in SED group(P < 0.05).2.Blocking the Wnt/β-catenin pathway could attenuate the protective effect of medium intensity running on cartilage: After 8 weeks of medium intensity running and intra-injection DKK1,the Makin’s score was significantly higher than that of MIR group(P=0.024),and higher than that of SED group.The cartilage thickness in MIR group was significantly higher than that in SED group(P=0.008)and MIR +DKK1group(P=0.032).The expression of ACAN m RNA in MIR and SED group was significantly higher than that in MIR+DKK1 group,and the expression of ADAMTS5 m RNA and COL2ɑ m RNA in MIR group were significantly higher than that in SED and MIR+DKK1 group.3.Blocking the Wnt/β-catenin pathway can change the bone remodeling of subchondral bone after medium intensity running: BMD of tibia and femoral subchondral bone plate in MIR +DKK1 group was significantly lower than that in SED and MIR group,Tb Th was significantly lower than that in MIR group,and porosity of subchondral bone plate of tibia was significantly increased.BMD and BV/TV of medial femoral subchondral cancellous bone in MIR +DKK1 group were significantly higher than those in MIR group,Tb.Sp and DA were significantly higher than those in SED group,and CD of lateral femoral subchondral cancellous bone in MIR+DKK1 group was significantly higher than that in MIR group.In tibial subchondral cancellous bone,BMD and BV/TV in MIR+DKK1 group were significantly lower than those in SED group,and Tb Th was significantly lower than those in SED and MIR group.Lateral BV/TV in MIR+DKK1 group was significantly lower than that in SED group,and Tb Th was significantly lower than that in MIR group.The toluidine blue staining results showed that the Ob.S/BS,N.Ob/T.Ar and N.Ob/B.Pm in SED and MIR groups were significantly higher than those in MIR+DKK1 group(P<0.05).The expression levels of ALP and CTSK were significantly higher than those of SED and MIR +DKK1,while the expression levels of ALP were significantly lower than those of SED and MIR+DKK1.Conclusion: Medium intensity running has a protective effect on cartilage by activating Wnt/β-catenin signaling pathway in subchondral bone.Chapter 3 Mechanism of infrapatellar Fat Pad /cartilage and subchondral bone/cartilage interaction in patients with osteoarthritis based on extracellular metabolomicsObjective: To investigate the interaction of infrapatellar Fat Pad /cartilage and subchondral bone/cartilage and their related mechanisms by using the metabolomics method.Methods: Fat conditioned media(FCM)of infrapatellar Fat Pad and bone conditioned media(BCM)of subchondral bone from patients with osteoarthritis were used to treat human OA chondrocytes,and the extracellular metabolites of human osteoarthritis chondrocytes were detected at different time points by non-targeted metabolic footprint analysis based on liquid chromatography and mass spectrometry(LC-MS),to find the different metabolites,and to explore the main metabolic pathways combined with bioinformatics methods.Results: 1.FCM could inhibit the proliferation of human OA chondrocytes:After treated with FCM for 48 h,the proliferation of human OA chondrocytes was slowed down,and FCM had a certain inhibitory effect on the proliferation of human OA chondrocytes(P=0.023).2.FCM and BCM can significantly affect the metabolic footprint of human OA chondrocytes,and FCM has a greater disturbance on the metabolic footprint of OA chondrocytes: on the pattern diagram of principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),after FCM and BCM treatment,,the data sample areas are obviously separated,indicating that FCM and BCM can significantly affect the metabolic footprint of human OA chondrocytes.Compared with OA + BCM group,OA + FCM group was more deviated from OA group,which indicated that FCM had more disturbance on metabolic footprint of OA chondrocytes.By metabonomics identification,131 different metabolites were screened after FCM treatment compared with before treatment.There were 19 metabolic pathways involved in the different metabolites between the two groups,including alanine,aspartate and glutamate metabolism,oxidative phosphorylation,glucagon signaling pathway,phenylalanine metabolism,c AMP signaling pathway,TCA cycle,etc.A total of 196 metabolites were screened after BCM treatment,there were 16 metabolic pathways involved in the different metabolites between the two groups,including glycine,serine and threonine metabolism,taurine and low taurine metabolism,nicotinic acid and nicotinamide metabolism,arginine and proline metabolism,cysteine and methionine metabolism,TCA cycle,etc.Conclusion: 1.Both FCM and BCM significantly affect the metabolic footprint of human OA chondrocytes,especially FCM.2.Infrapatellar fat pad aggravates OA chondrocyte injury by changing the metabolism of amino acids,pyrimidine and purine in chondrocytes and up regulating TCA cycle.3.Subchondral bone affects articular cartilage by changing the metabolism of amino acids,pyrimidine and purine in OA chondrocytes,up regulating the levels of taurine and nicotinamide,and participates in multiple functions such as chondrocyte repair and chondrocyte injury. |
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