The Influence And Mechanism Of Different Stress On Knee Articular Cartilage Metabolism Of Rats In Vivo

Objective: Biomechanical factors play an important role in the pathophysiology of osteoarthritis(OA).Our research group has designed a novel rat running equipment,which can be used to achieve semi-quantitative of mechanical stimulation by slope,to gain rat OA model.Then we will study its tissue and gene expression level changes in the process of the occurrence and development of OA,to explore the important role of mechanical stimulation in the pathophysiology of OA and related mechanisms in vivo.Methods: 84 Sprague Dawley(SD)rats,about 3 months old,were randomly assigned to each group.The mechanical stress of the rat knee joint was semi-quantitative measured by different slopes,such as 0,10 and 20°.The groups of long-time running protocol should load running scheme for 30 d,at a speed of 1km/h and duration of 1h/d.After that,the rats were sacrificed by cervical dislocation method,and bilateral hind knee joints cartilage were taken for subsequent operation.While the groups of acute running protocol did the exercise for only once(running speed of 1km/h for 1h).Bilateral hind knee joints cartilage were also taken at time points of 0h,2h,6h,12 h and 24 h after acute running training,respectively.Qualitative observation of articular cartilage morphology and aggrecan(AGG)content change were surveyed after HE,safranin O and toluidine blue staining,and differences of quantitative evaluation of cartilage injury between each group were assessed according to the OOCHAS cartilage injur y semi-quantitative evaluation score system.Type II collagen,type X collagen,IL-1β,TNF-α,MMP-3,MMP-9 and MMP-13 expression of cartilage tissue in each group were studied by immunohistochemical method.As well,total m RNAs were extracted from chondroc ytes and then reverse transcripted to c DNAs,which were detected by RT-PCR to illuminate gene expression levels of type II collagen,AGG,type I collagen,type X collagen,IL-1,IL-6,MMP-3,MMP-9,TNF-α,MMP-13,SOX9 and ADAMST5 in the cartilage.SPSS software was used for the analysis of variance and the comparison between the groups,and the test level was P < 0.05.Results: In the rats with long-term stress running protocol,there were different degrees of damage to the articular cartilage at the end of the experiment.Compared with normal cage control group,qualitative observation result of HE staining,safranin O and toluidine blue staining in rat knee cartilage tissue of panel treadmill running group was not significantly different.While,in 10° and 20° uphill treadmill running groups,the content of AGG in the articular cartilage was decreased gradually as well as the safranin O and toluidine blue staining of rat knee cartilage.And there were different degrees of visible articular surface injury,as well as damage degree of 20° uphill treadmill running groups was higher than 10°.Furthermore,the chondrocytes showed hypertrophy performance in a certain extent,such as reduced chondrocytes number,increased cytoplasm scale and significantly larger cell lacuna,disorganized cell order,lost typical hierarchical structure of articular cartilage,significantly thinner cartilage thickness,tide line disappeared or became unobviously.With the uphill exercise angle increasing,the stress load to what the rats knee joint is subjected was increasing.The analysis showed that the OOCHAS score of the knee articular cartilage increased along with the increasing stress loading using one way ANOVA(F=122.4,P < 0.0001)and differences between groups were statistically significant by using S-N-K method(P < 0.05),and indicating that tissue injury accentuated gradually.Immunohistochemistry results showed that,with the running gradient(i.e.stress load)increasing,the content of type II collagen in articular cartilage decreased gradually,while the content of type X collagen gradually increased when compared with the control group.Tissue expression of inflammatory mediators,such as IL-1β,TNF-α,MMP-3,MMP-9 and MMP-13,were increased in different degrees too.Our RT-PCR results also showed that the gene expression level of rat articular cartilage type II collagen and AGG m RNA decreased gradually,while IL-1,TNF-α,MMP-3,MMP-9 and MMP-13 m RNA expression level increased significantly along with the increasing of the stress load when compared with the control group.Meanwhile,increase of TNF-αand MMP-3 was the most obviously.Overall,the increase of expression level of IL-1 was not so obviously,and there was no obviously different of IL-1 m RNA expression between 10° uphill running group and panel group.Rats in acute stress running protocol were just taken once running exercise.Then gene expression levels of the m RNAs were detected by RT-PCR method after running.The expression of type II collagen appeared to increase temporarily at 6h,then returned to normal levels,and positively correlated with stress intensity.The expression level of AGG,COL I,COL X began to gradually increase from 6h,and it was significantly increased at 24 h.IL-6 was the fastest increased gene after the uphill exercise,then gradually decreased;IL-1 and TNF-α expression began to increase from 12 h,but the overall expression was not so obviously.The expression of MMP-3,MMP-9 and MMP-13 began to increase from 6h,and the uphill running group was significantly higher than those of control group and panel running group at 24 h,and the increase of MMP-13 was the most obviously,with nearly 10 times higher expression level of 20° uphill running group at 24 h.Gene expression of SOX9 slightly increased at early stage.After that,there was no difference between the uphill running groups and the control group.The gene expression of ADAMST5 reached the highest level at 2h,then gradually came back to normal level.All in all,the change amplitude of most gene expression increased with the stress intensity.Conclusion: Long-time excessive stress loading on rat knee cartilage articular can induce cartilage degeneration,reduce the number of chondrocytes,result in hypertrophy changes associated with chondrocytes,damage the cartilage matrix and lead to degradation of AGG,reduce the content of type II collagen,increase the content of type X collagen.The articular cartilage degeneration will come up,so that the friction resistance of the articular surface increases and its mechanical properties can no longer adapt to joint activities.Articular cartilage will be teared and weared under the load of excessive stress,and knee OA will occur ultimately.This change should be positively related to exce ssive intensity of the power load.Excessive stress may activates IL-1 and TNF-α by increasing the expression level of IL-6 firstly,then promotes the m RNA synthesis of MMPs family,and leads to the degradation of the cartilage matrix.While the increase of SOX9 and ADAMST5 gene expression may be a cause of chondrocytes hypertrophy and apoptosis.