The Antioxidative Effect Of Peroxiredoxin Ⅰ In Osteoarthritic Cartilage

BackgroundOsteoarthritis (osteoarthritis, OA), as the most common joint disease, which leads to disability is the second highest incidence diseases over the age of50-year-old. The pathological features of OA shows progressive cartilage degeneration, cracked and defect, and cartilage is lack of self-healing capability. Now the key point about the treatment of OA is to find the cause of OA progresses, but not conservative treatment.In recent years, the pathogenesis of OA is not yet entirely clear, though the academic did a lot of studies from different angles. Apoptosis is considered to be a key pathological factor for cartilage degeneration. Mollenhauer and Mok reported: apoptotic cells were usually in the surface of OA cartilage or around cartilage defect lesions. OA cartilage defects were mostly present in the cartilage surface, the difference distribution of apoptotic cells during the abnormal layers is considered to be the key reason not to repair the cartilage defects.In the joint cavity of OA patients, there is high concentrations of active oxygen disproportionation material (reactive oxygen spesies ROS). The more attention is the free radical theory which scholars have put forward, such as ROS will lead to membrane lipid oxidation and lipid oxidation metabolites increasing which will directly attack chondrocyte DNA, affect DNA synthesis, eventually lead to apoptosis. Loeser and Carlson found that ROS could make the proteins of intracellular nitration which existed in the superficial of the cartilage tissues of OA, that indirectly reflects the distribution of ROS being different during the cartilage tissues. There may be some relationship between ROS aggregating in OA superficial tissues and the apoptosis of chondrocytes also emerging in the same layer.Just some scholars reports that there were the different distributions of OA cartilage tissue, so we take more attention about the antioxidant enzymes family peroxiredoxins (Prxs) which are the scavenger of ROS. there are at least six Prx subtypes(Prx Ⅰ-Ⅵ)in mammals they can be divided into three subcategories:①2-Cys(Prx Ⅰ-Ⅳ);②atypical2-Cys(Prx Ⅴ);③1-Cys(Prx Ⅵ). All of the three type proteins have the same cysteine which plays biological effect. Literature reviews repord that Prxs belong to peroxidase family, they use electrons which is from thiol protein to restore oxygen free radicals into non-toxic substances. Because of its unique structure and role, more and more people particularly pay attention to the study about Prxs which can reduce and deradate hydroxide, peroxynitrite, and many kinds of organic hydroperoxides (ROOH+2e-�'>ROH+H20). Prxs widely exist in variety of vivo, its oxidation is similar to glutathione peroxidase (glutathione peroxidase the GPX) and catalase (catalase, Cat). Current study repords that the Prxs family are widely distributed in the cells, the functions they play in are that as antioxidant, promoteing cells proliferation and regulating intracellular signal transduction. As the scavenger of ROS, Prxs that are different in the distribution of OA tissue play what role, which paid all of us worthy of attention.We found differential proteins between the normal and OA cartilage tissue by two-dimensional gel electrophoresis and mass spectrometry, One of them is Prx I which belongs to the peroxiredoxins family(Prxs). In theory, the high expression of Prx I can take the capadity of anti-apoptotic, but why it does’t do that? Whether there are more Prx I in the cartilage surface where ROS enrichs in? That view is the focus. So we plan to explore this area.In our experiment, The overall expression level difference of Prx I was compared by Western blot and Prx I zonal expression inside the normal and osteoarthritic cartilage was detected by immunohistochemistry, the same time knocking-down Prx I in the cartilage cells simulate the phenomenon of downregulation of Prx I in the surface of OA cartilage, and explore the relationship between the lower expression of Prx I and OA.ObjectivesTo observe the differential expression and distribution of Prx I expression in normal and OA cartilage and knock-down Prx I in the cartilage cells to simulate the phenomenon of downregulation of Prx I in the surface of OA cartilage, the aim is to explore the relationship between the lower expression of Prx I and chondrocytes anti-apoptotic ability to ROS and at last to explore the causes that OA cartilage be lack of self-healing ability.Methods1. The expression and distribution of Prx I in normal and OA cartilage tissue(1) Study the expression and distribution features of Prx I difference in normal and OA cartilage by western blot;(2) observe the expression and distribution characteristics of Prx I in normal and OA cartilage tissue by immunohistochemical staining.2. Isolation and culture of human chondrocytesCulture chondrocytes by two primary methods of enzyme digestion and enzyme pre-digesting tissue attachment-block, while identify the collagen Ⅱ by immunohistochemical.3. Explore whether Prx I could prevent from oxidative damage in chondrocytes(1) Use the Primer3.0software design Prx I siRNA and seclect the best three to biosynthesis(Sequence A:5’-GAU GGU CAG UUU AAA GAU ATT-3’; B:5’-GCC GAA UUG UGG UGU CUU ATT-3’; C:5’-GGG UCA AUA CAC CUA AGA ATT-3’). then transfected siRNA with LipofectamineTM2000to knocking-down Prx I in the second generation cultured human chondrocytes, and after72h filter the siRNA which has the best knocking-down efficiency by Western blot. And using the the best siRNA to transfect chondrocytes, then extract chondrocytes soluble protein at the time Oh,24h,48h,72h,96h,120h and observe the expression level of Prx I at the different time points to confirm the best knocking-down efficiency time.(2) Chondrocytes were randomly divided into normal group and knocking-down Prx I group, then at the best knocking-down efficiency time simulate chondrocytes of two groups by0.6mM H2O2for Omin,90min,180min, then extract chondrocytes soluble protein and detect early apoptosis indicators:cleaved-caspase3and the procaspse3by WB. at the same time simulate two groups chondrocytes by0.6mM H2O2for Omin,30min,90min,180min, then annexin V/PI dual stained normal group and knocking-down Prx I group cells, then detect the apoptosis of the two groups cells by flow cytometry. And explore the relationship between the lower expression of Prx Ⅰ and chondrocytes anti-apoptotic ability to ROS.Result1. The expression and distribution of Prx I in normal and OA cartilage tissue(1) WB results show that the relative expression level of Prx I protein is1.014±0.189and3.893±0.693, that OA cartilage tissue is2.89-fold(t=18.34, P<0.05) higher than normal cartilage tissue.(2) Immunohistochemical staining showed that the expression and distribution of Prx I in superficial, middle and deep layer is relatively uniform. Prx I colored light brown-yellow granules in the cytoplasm, but no positive color in the nucleus and cytoplasm. Moderate OA cartilage tissue, cartilage surface showed varying degrees of wear, the cells did not keep the normal arrangement, but the cell morphology in middle layer is relatively normal and tide line of the deep layer cells And the expression of Prx I in the deep layer cartilage cells significantly increased and cytoplasmic generally stained yellow, but the abundance of Prx I in middle layer is weaker than the deep layer, the superficial layer have a few expression. In severe OA cartilage tissue, the surface cartilage wore seriously, showed different degrees of fibrosis and cracks and there were cell clusters. But, tide line structure tend to disappear in deep layer. Each layers is more obvious different in severe OA cartilage. the majority cells dyed weak or even absence in surface.However, in the middle and deep layer there are dark brown-yellow granules in the entire cell contour except the nucleolu, while there was cloudy staining around the chondrocytes.2. Isolation and culture of human chondrocytesEnzyme digestion isolated cartilage cells which usually adhered to culture flask in12-24h, primary cells distributed uniformly, like spindle-shaped, polygonal and abundant cytoplasm; round nuclei habitat in the central. We will see around cells on tissue block by enzyme pre-digestion culture, some cells began to stretch, around the block no cell climbed out in2-3d. After4-5d, cells moved out around some block, and cells grow radially and cell morphology was spindle and polygonal. The4th chondrocytes began to appear the phenomenon of dedifferentiation, cell antennae began to increase, cell volume increased, the cytoplasm and the particles increased, nucleolar hypertrophy, cell ill-defined often overlap. One week after the primary chondrocytes cultured collagenII staining were brown positive.3. Explore whether Prx I could prevent from oxidative damage in chondrocytes.(1) siRNA labeled by Carboxyfluorescein (FAM) transfect primary chondrocytes, there were more than85%-90%of the cells being fluorescent observed under fluorescence microscope after4h. transfection efficiency can meet the requirements of the experiment.(2) WB filter the highest transfection efficiency siRNA by WB from the A, B, C siRNA. FAM-labeled siRNA transfect primary chondrocytes and after72h the relative expression level of Prx I protein is0.778±0.498,0.318±0.300and0.223±0.129. three groups expression level of Prx I is lower by22.2%(F=459.858, P<0.001),68.2%(F=459.858, P<0.001) and77.7%(F=459.858, P<0.001) than the NC group, it showed that C siRNA had the best efficiency knocking-down Prx I.(3) We used the Prx I C siRNA to knockdown Prx I, after24h,48h,72h,96h,120h the relative expression level of Prx I protein is0.837±0.222,0.531±0.015,0.214±0.016,0.314±0.029and0.392±0.020, five groups expression level of Prx I is lower by16.3%(F=746.383, P<0.001),46.9%(F=746.383, P<0.001),78.6%(F=746.383, P<0.001),68.6%(F=746.383, P<0.001) and60.8%(F=746.383, P<0.001) compared with the negative control group, it showed that the relative expression level of Prx I protein is the lowest after knocking-down Prx I for72h.(4)The relationship between knocking-down Prx I of cartilage cells and anti-apoptotic abilitythe observations showed:the longer time, the worse of the degree of apoptosis of two groups. knocking-down gene group was worse than the normal group stimulating the same time. Under the microscope, two groups cells keep good condition stimulating for Omin. while the cells are no longer spindle but into circular and inclusions significant contracted and membrane appears hole, nuclear condensation, cell density significantly decreased, part of the cells apoptosis and suspended in the medium, while the normal group stimulating for180min is significantly better.Western blot analysis:using0.6mM H2O2to stimulate the control group chondrocytes for90min and180min, aprocaspase3protein relative expression are0.563±0.017and0.326±0.015, compared with NC group, the two time points reduced by43.7%(F=2310.82, P<0.001) and67.4%(F=2310.82, P<0.001). but the relative expression of cleaved caspase3protein gradually increase with the stimulation time extended. Stimulating for90min and180min, they are1.218±0.093and4.264±0.118, increased by21.8%(F=2981.36, P>0.05) and326.4%(F=2981.36, P<0.001) than NC group. the knocking-down group chondrocytes for90min and180min, aprocaspase3protein relative expression are0.541±0.011and0.173±0.012compared with NC group, the two time points reduced by42.9%(F=2310.82, P<0.001) and82.7%(F=2310.82, P<0.001),. but the relative expression of cleaved caspase3protein gradually increase with the stimulation time extended. Stimulating for90min and180min, they are5.543±0.091and7.761±0.1048, increased by454.3%(F=2981.36, P<0.001) and676.1%(F=2981.36, P<0.001) than NC group. At the time90min and180min, the relative expression of procaspase3protein of knocking-down group reduced by3.9%(t=-0.715, P=0.51) and46.9%(t=13.177, P<0.05), And the relative expression of cleaved caspase3protein of knocking-down group incresed by355.1%(t=-57.332, P<0.05) and82.0%(t=-38.322, P<0.05).Annexin V/PI staining cartilage cells, using0.6mM H2O2to stimulate the control group chondrocytes for Omin,30min,90min,180min, flow cytometry detected the sum of the early and late stage apoptosis rate for the control group, the ratio are4.72%±1.05%,10.43%±1.10%,20.14%±0.89%,30.30%±0.95%. but the knocking-down Prx I group is4.83%±0.89%,16.53%±0.90%,30.62%±1.10%,68.54%±1.24%. Stimulating the cells for90min and180min, the control group and the knocking-down group showed significant difference, the sum of the early and late stage apoptosis rate of the knocking-down group increased by52.0%(t=12.779, P=0.03) and126.2%(t=-42.195,P=0.14) compared with the NC group.Conclusions(1) In this study, we verified the function of Prx Ⅰ in the pathogenesis of OA from the tissue and cell level. In cartilage tissue, the expression of Prx I in OA cartilage tissue was significantly higher2.89fold than normal cartilage tissue; Prx I is more evenly distributed in normal cartilage, but less expression in the superficial, layers of OA cartilage, higher in the deep layer.(2) The lower expression of Prx I can not effectively prevent the oxidation of ROS from stimulating and there are more apoptosis cells in the superficial layer where the expression of Prx I is lower.