| Osteoarthritis (OA) is the most common disease in orthopedics. World Health Organization (WHO) lists OA, cardiovascular diseases and cancer as "the three major killers" to human health. Coming along with the early arrival of the aging of population, incidence of OA is rising year by year. OA has become a serious public health problem due to the lack of effective measures which could greatly improved the state of the illness. OA’s main characteristic change is the progressive degeneration of articular cartilage, causing pain and leading to joint dysfunction ultimately. There is still no clear conclusion of the specific mechanism of cartilage degeneration, but the extensive research suggests that the cartilage degeneration began in the chondrocytes, which is the only cell in cartilage. Chondrocytes apoptosis and the declining synthesis ability of extracellular matrix (ECM) are two important symbols of cartilage degeneration. Several factors can cause chondrocytes apoptosis or declining synthesis ability, however, the abnormalities of the cell energy metabolism can induce chondrocytes apoptosis and the declining synthesis ability at the same time.As an important organelle in cell, the main function of mitochondria is to maintain the balance of energy metabolism in cells, lower ROS to avoid oxidative stress, against the damage of inflammatory, etc. And the function of mitochondria is mainly regulated by the proliferator-activated receptor y coactivator 1α (PGC-1α). According to the studies at home and abroad, mitochondrial synthetic dysfunction and PGC-la abnormal expression exists in a variety of degenerative disease and age-related diseases; the activity and expression of PGC-la are regulated by AMPK and SIRT-1 in a variety of cells; and study also confirms that AMPK and SIRT-1 regulate and protect the cell apoptosis mediated by inflammatory factors IL-1β、IL-6、 NF-κB. From what has been discussed above, we speculate that mitochondrial dysfunction synthesis and energy metabolism mediated by AMPK/SIRT-1/PGC-1α may exists in OA cartilage and the reduction of ECM level and chondrocytes degeneration also has close ties with them. Therefore, combined with domestic and foreign related research foundation, this system research will lead to explore the function mechanism of AMPK/SIRT-1/PGC-1α in human articular cartilage and chondrocytes in rats.Part Ⅰ:the expression of AMPK/SIRT-1/PGC-la in human articular cartilage of osteoarthritis and correlation researchObjective:The purpose of this study is to observe the expression of AMPK/SIRT-1/PGC-1α in articular cartilage of osteoarthritis and discuss the effects of this signal pathway on human cartilage extracellular matrix synthesis ability.Methods:We collected knee joint cartilage from patients with knee arthroplasty and amputation for various reasons. Amputation surgery patients get control of the lateral femoral condyle, knee arthroplasty surgery resection have been the knee arthritis, according to the parts and specimens of relative weight bearing area is divided into relatively non-loaded part and relatively loaded part. We used HE and Safranin O staining to observe cartilage morphology; Mankin assessment to assess the degree of cartilage degeneration; Real-time PCR to detect cartilage Aggrecan and Collagen type-2 mRNA; Western blot to detect AMPK, SIRT-1 and PGC-1αof cartilage.Results:HE staining and Safranin O staining were observed that degeneration of relatively loaded part was serious than relatively non-loaded part, such as, chondrocyte hypertrophy, proteoglycans and collagen loss, blurred tide line disappeared, like the canal changes, and with enhanced degeneration aggravated. Mankin scores gradually increased in the control group, non-loaded part, loaded part (P<0.05). Real-time PCR assay showed Aggrecan and Collagen type-2 mRNA expression level was significantly decreased (P<0.05), which is consistent with the results of Mankin scores. RT-PCR and Western blot showed that AMPK, SIRT-1, PGC-1α expression levels in three groups of cartilage tissue also decreased (P<0.05), and were relevant with the Mankin score, proteoglycans, type Ⅱ collagen mRNA expression levels.Conclusion:The normal morphological of human articular cartilage changes disappeared, chondrocytes was degeneration and necrosis, the tide line was blurred or even disappear, down proteoglycans and type Ⅱ collagen expression levels; within AMPK, SIRT-1, PGC-1α mRNA and protein levels of OA cartilage were significantly reduced, and the tendency to reduce was positively correlated with cartilage degeneration, suggesting that human articular cartilage degeneration of the knee change may be related to downregulation of AMPK, SIRT-1, PGC-1α.Part Ⅱ:The effect and mechanism of PGC-1α on mitochondrial synthesis of chondrocytes of rats in vitroObjective:The purpose of this study is to research the effect and mechanism of PGC-1α on mitochondrial synthesis of chondrocytes of rats in vitro.Methods:We used ACLT to establish OA mode in rats, contralateral sham surgery as the control group. Chondrocytes from both sides of the knee cartilage were isolated and cultured. We successfully constructed PGC-1α siRNA plasmid and transfected into chondrocytes and established stably expressing cell lines. So there were four groups of chondrocytes:normal control group, OA group, PGC-1α siRNA group and OA+PGC-1α siRNA group. We used western blot to detect the PGC-1α, NRF1, NRF2, TFAM in all groups; luciferin-luciferase assay to detect ATP content in all groups; BCA assay to detect the activity of chondrocytes mitochondrial respiratory chain complex Ⅰ, Ⅲ; FCM to detect apoptosis in all groups; RT-PCR to detect Aggrecan and Collagen type -2 mRNA expression levels in all groups.Results:Western blot showed that:PGC-1α expression in OA group, PGC-1α siRNA group, OA+PGC-1α siRNA group was significantly reduced, corresponding NRF1, NRF2, TFAM were downregulation (P<0.05). Mitochondrial respiratory chain complexes Ⅰ, Ⅲ activity and ATP content in chondrocytes showed that:OA group, PGC-1α siRNA group, OA+PGC-1α siRNA group were significantly lower (P<0.05), hinted mitochondria biosynthetic functions are impaired. FCM analysis showed that: apoptosis in OA group, PGC-1α siRNA group, OA+PGC-1α siRNA group were significantly increased (P<0.05). RT-PCR showed that:Aggrecan and Collagen type-2 mRNA expression in OA group, PGC-1α siRNA group, OA+PGC-1α siRNA group were significantly reduced (P<0.05).Conclusion:Downregulation of PGC-1α in Chondrocytes of rats will lead to downstream regulator NRF1, NRF2, TFAM downregulated, the mitochondrial biogenesis function was reduced and damaged, the impairment of cellular energy metabolism occured, which were leading to increased apoptosis, extracellular matrix loss and cartilage degeneration. As OA chondrocytes were downregulation of PGC-1α and the downstream regulatory factors NRF1, NRF2, TFAM, showed mitochondrial biogenesis dysfunction phenomenon, we speculated that PGC-1α, NRF1, NRF2 and TFAM related signaling pathways downturn may be an important part of the process of OA.Part Ⅲ:The role of AMPK/SIRT-1 in mitochondrial synthesis of chondrocytes of rats in vitroObjective:The purpose of this study is to research the effect and mechanism of AMPK/SIRT-1 on mitochondrial synthesis of chondrocytes of rats in vitro.Methods:We use AMPK activator AICAR, AMPK inhibitor Compound C, SIRT-1 activator Resveratorl, SIRT-1 inhibitors EX527 respectively pretreated normal and OA chondrocytes of rats, to observe AMPK and SIRT-1 activity changes in chondrocytes. Respectively, we use the specified test kit in each group chondrocytes to detect the activity of AMPK and SIRT-1; RT-PCR to detect mRNA expression levels of PGC-la, NRF1, NRF2, TFAM; BCA method to decet mitochondria respiratory chain complexes Ⅰ, Ⅲ activity; luciferin-luciferase to measure ATP content; FCM to detect apoptosis; RT-PCR to detect mRNA expression of Aggrecan and Collagen type-2.Results:The results showed that:the AMPK and SIRT-1 activity in AICAR group and Resveratorl group was significantly higher than the control group and the OA model group (P<0.01), corresponding, the AMPK and SIRT-1 activity in Compound C group, EX527 group and OA group was significantly decreased than the control group (P<0.05). AICAR and Resveratorl also significantly enhanced AMPK and SIRT-1 activity in OA chondrocytes (P<0.05). RT-PCR showed that:AICAR and Resveratorl can significantly enhance the mRNA expression levels of PGC-1α and downstream regulatory factors NRF1, NRF2, TFAM both in the control group and OA group (P<0.05). The opposited, Compound C and EX527 can inhibit mRNA expression levels of these indicators (P<0.05). BCA method and the luciferin-luciferase assay showed that:AICAR and Resveratorl can significantly enhance the respiratory chain complexes Ⅰ, Ⅲ activity and intracellular ATP levels both in the control group and OA group (P<0.05); Compound C and EX527 significantly inhibited the respiratory chain complexes Ⅰ,Ⅲ activity and intracellular ATP levels(P<0.05). FCM and RT-PCR results showed that:AICAR and Resveratorl can significantly reduce the apoptosis rate of control group and OA group (P<0.05), significantly enhanced the mRNA expression levels of Aggrecan and Collagen type-2 in the normal chondrocytes(P<0.05), but no significant increase in OA group (P>0.05); Compound C and EX527 significantly increased the apoptosis rate of control group and OA group (P<0.05), significantly inhibited Aggrecan and Collagen type-2 mRNA expression levels in both control group and OA group (P<0.05).Conclusion:Chondrocytes of rats existed positive feedback of AMPK interaction with SIRT-1, and both decreased in OA chondrocytes. This reduction is likely to affect the synthesis of mitochondrial function, intracellular energy metabolism, apoptosis and extracellular matrix synthesis in chondrocytes phenotypic changes by PGC-la and the downstream regulatory factors NRF1, NRF2, TFAM. Enhancing the activity of AMPK and SIRT-1 in chondrocytes may an important target of delaying or curing osteoarthritis by inhibiting apoptosis and enhancing extracellular matrix synthesis. |
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