| Background:Osteoarthritis (OA) is the most common disease in old people. The typical pathological changes in OA is degradation of articular cartilage, including fibrosis, attenuation of cartilage, mussy structure of chondrocyte and the decrease of cartilage matrix. Extracellular matix is consist of proteoglycan and collagens, the imbalance in anabolism and catabolism contributes to the loss of articular cartilage. In previous studies indicated that cytokines as IL-1β、IFN-y and TNF-a which could suppress the synthesis of aggrecan, the most abundant proteoglycan in the extracellular of chondrocytes. Therefore, inflammatory factors played an important role in the process of OA.IL-18was initially thought to IFN-y induced factor. The structure of IL-18is similar to IL-1. In normal condition, many kinds of cells including Kupffer cells, macrophages, keratinocytes, dendritic cells, astrocytes, microglia, respiratory epithelial cells, osteoblasts and chondrocytes can secrete inactive precursor of IL-18, the pro-IL-18. Like IL-1, IL-18is synthesized as inactive precursor, and decomposed to muture IL-18by interleukin1β converting enzyme (ICE, also called Caspase-1). Oxidative stress and lipopolysaccharide binding protein stimulate the Caspase-1signaling pathway followed by increasing largely of active IL-18. On the one hand, IL-18can appeare biologic activity as the assistant factor of heper T cell lor2, on the other hand, IL-18will induce the product of Fas ligand, matrix metalloproteinase (MMPs) and some other inflammatory factors. IL-18also contributes to some disease, including intestinal diseases, actinic dermatitis, asthma, chromic fibrous pneumonia and chronic obstructive pulmonary disease by promoting expression of a series of inflammatory factors, such as interferon-gamma (IFN-y), granulocyte colony stimulating factor (G-CSF), TNF-a and IL-1β.It is noticeable that IL-18plays an important role in rheumatoid arthritis (RA). Studies demonstrated the existence of IL-18in the synovial fliud of RA patients. And IL-18receptor (IL-18R) was also found in the synovium and synovial fluid of OA patients. Decrease of macrophages in synovium will inhibit the degradation of cartilage. Furthermore, IL-18was observed by immunohistochemical staining, and the factor would promoted the apoptosis of chondrocytes.In our prvious studies, the data showed that the concentration of IL-18was related to that of PGE2in the supernatant of cultured synoviocytes. Compared with normal synoviocytes, OA synoviocytes produced higher level of IL-18. However, the effects of IL-18on synoviocytes, chondrocytes and degradation of cartilage is still unknown. At present study, the purposes were to investigate the pro-inflammatory role of IL-18on OA synoviocytes and chondrocytes, and the edffect of IL-18on articular cartilage.Objective:1. To investigate the effect of IL-18on the proliferation of chondrocytes.2. To investigate the role of IL-18on the inflammatory of human chondroctyes and synoviocytes. 3. To establish the OA model initially with IL-18to investigate the effect of IL-18on the cartilage in rats’knee joints.Methods:1. Isolation and culture of OA chondrocyte.Chondrocytes from femur condyle and tibial plateau in OA patients who received total kee joint replacement. The samples were collected and cut into0.5-1mm3slices. Then were digested at37℃on a shaking table in DMEM containing0.2%collagenase Ⅱ and0.1%hyaluronidase for6h. Cells in supernatant were filtered by a sterile, plated in25cm2culture flasks, then incubated in a humidified atmosphere containing5%carbon dioxide (CO2). Cells were passaged when reached80%confluence. The chondrocytes before second passage were used for the following research.Inverted contrast biological microscope was used to observe the pattern of chondrocytes and the images were collected and analyzed. Chondrocyte were inoculated in to96-well plates. MMT method was used to detect the cell proliferation and the growth curve was described. Also collagen II of chondrocytes were stained by immunohistochemistry.2. Isolated and culture of human synoviocytes in kee joints. The synovial tissue was washed twice using sterile PBS. The sample was cut into sections digested with0.1%trypsin at37℃for30min. The tissue was then placed in DMEM containing0.1%type I collagenase and10%fetal bovine serum (FBS) for2h. After filtration and centrifugation, the cells were collected and cultured in medium with penicillin and streptomycin at37℃in a5%carbon dioxide environment. Also collagen I and VCAM-1were respectively detected by immunohistochemistry and FCM.3. The effect of rhIL-18on chondrocytes and synoviocytes. (1) The effect of rhIL-18on the cell proliferation.Chondrocyte were inocubated into96-well plate and incubated with different concentration of rhIL-18(0,50,150,300ng/ml), TGF-β (50ng/ml) and IL-18+TGF-β for48h. MTT method was used to exam the cell proliferation.(2) Cell preparation and RT-PCRChondrocyte were inocubated into96-well plate and incubated with different concentration of rhIL-18(0,50,150,300ng/ml) for48h. Equivalent G1, G2and G3synoviocytes were starved in DMEM for24h, then cultured with IL-18(10mM/ml). The total RNA was extract to exam the mRNA expression of inflammatory factors, collagen Ⅱ and aggrecan in chondrocytes using reverse transcription polymerase chain reaction (RT-PCR).(3) Detecton of PGE2, TNF-α and MMP-13in supernatantChondrocyte were inocubated into96-well plate and incubated with different concentration of rhIL-18(0,50,150,300ng/ml) for48h. Equivalent G1, G2and G3synoviocytes were starved in DMEM for24h, then cultured with IL-18(10mM/ml). Collect the supernatant. Then enzyme-linked immunosorbent assay (ELISA) was adopt to explore the level of PGE2, TNF-α and MMP-13.(4) RT-PCR was used to detect the express of IL-18receptors in chondrocytes and synoviocytes.4. The effect of rrIL-18on the knee joint degeneration in SD rat(1) Groups and intervene20SD rats received received intra-articular injection of rrIL-18(200ng) diluted with phosphate buffer saline (PBS) in their left knees thrice a week as experiment group. On the right knees, as control group, the rats received equivalent PBS. Half samples were collected and detected after6w and12w, respectively. (2) X-ray of rats’knee joints.After the rats were anesthetized and fixed on cystosepiment with limbs binding on a series pins, X-ray detection was used to evaluate the degeneration of the knee joints in control group and experiment group.2independent observers were ask to judge the knee degeneration.(3) Histopathology test of cartilageSacrifice the rats and collect the knee joints. After fixated in paraformaldehyde, decalcification and paraffin-embeded, the samples were cut into5μm slices and stained with stained with Safranin O/Fast green. The images were collected and digitized. Cartilage area were analyzed by Image-Pro Plus.(4) Immunohistochemistry of cartilage.After fixated in paraformaldehyde, decalcification and paraffin-embeded, the samples were cut into5μm slices. Immunohistochemistry was take to exam the express of COX-2and aggrecan in cartilage.Results:1. Culture and identification of chondrocyte.(1) The primary chondrocytes show irregular shape. Most were radial, polygon and ellipse in the flask. While when after passaged3times, the chondrocytes became are generally elongated and spindle shaped.(2) The proliferation of chondrocytes. Over time, the number of chondrocytes was always growing (P<0.01) until entering the platform period after10days. At the same time, the cell number between passage1and2was not significantly different (P=0.939). And there was not interaction between time and passages (P=0.577).(3) Immunohistochemistry staining Immunohistochemistry staining suggested that collagen Ⅱ expressed in all the chondrocytes.2. Culture and identification of synoviocytes.(1) The pattern of synoviocytes. Type A and type B synoviocytes respectively appeared polygon and elongated and spindle shaped. With the passages increased, the rate of type B synoviocytes increased.(2) The proliferation of synoviocytes. Over time, the number of synoviocytes was always growing (P<0.001) until entering the platform period after4days. At the same time, the cell number between passage1,2and3was not significantly different (P>0.05). And there was not interaction between time and passages (P=0.987).(3) Immunohistochemistry staining and FCM detection Immunohistochemistry staining and FCM detection suggested that collagen Ⅱ and VCAM-1expressed in synoviocytes.3. rhIL-18induce the secretion of inflammatory factor and suppress the synthesis of cartilage matrix(1) Different concerntrations of rhIL-18(0、50、150'300ng/ml) appeared no significant effects on the number of chondrocytes (P>0.05). Compared with control group, cell number in TGF-β+IL-18group and TGF-β was increased significantly (P<0.001and P=0.016, respectively). And, the cell number in the TGF-β+IL-18group was significantly lower than that in TGF-β(P=0.002).(2) rhIL-18suppress the product of aggrecan and promote the express of inflammatory factor Compared with control group, the mRNA expression of aggrecan in300ng/ml group deceased significantly (P=0.008). However, mRNA of collagen II in every group expressed without any difference (P>0.05). rhIL-18could promote the secretion of COX-2and TNF-a in a dose-dependent manner. mRNA expression of COX-2in300ng/ml was higher than that in control group and150ng/ml group (P=0.006, P=0.036, respectively). While, compared with control group, the mRNA expression of TNF-a in150ng/ml group and300ng/ml increased significantly (P=0.001, P=0.023, respectively). Also, IL-18upregulated the mRNA expression of COX-2and TNF-a in G1synoviocytes.(3) Examination of the levels of PGE2, TNF-a and MMP-13in supernatant With the increase of rhIL-18, the levels of PGE2and TNF-a increased significantly (P<0.001). The concentration of PGE2in50ng/ml group appeared different with that in control and300ng/ml group (P=0.001, P=0.001, respectively). While incubated with different levels of rhIL-18, the content of MMP-13showed no significant differences (P>0.05). In Gl synoviocytes, levels of PGE2and TNF-a in IL-18were significant higher than than in control group (P=0.005, P=0.007).4. rhIL-18induce the cartilage matrix degradation and inflammatory reaction.(1) IL-18in rats’knee joints The concentrations of IL-18in experiment and control group were464.64±31.26pg/ml and15.26±1.62pg/ml. There is significant difference between them (P=0.002).(2) X-ray detection of samples At6-week,2joints appeared narrow joint gap or osteophytes in experiment group, while only1joint appeared narrow joint gap in control group (P>0.05). At12-week,5joints in the experiment group showed degeneration, no abnormal changed happened in control group. There is significantly difference in the degeneration rate between2groups (P=0.003).(3) Histopathology staining (Safranin O/Fast green staining) Area of femur condyle cartilage in control group was401134.62±119975.29mm2, and that in experiment group was345607.70±108555.66mm2. The latter significantly decreased compared with the former (P=0.035).(4) Immunohistochemistry At12-week, compared with control group, the expression of aggrecan in femur condyle cartilage in experiment group decreased (P=0.021). Also there is significant expression of aggrecan in tibial plateau between the2groups (P=0.047). Whatever in femur condyle or tibial plateau, the expression of COX-2in cartilage in experiment group was significantly higher than that of control group (P=0.002, P=0.032; respectively).Conclution1. IL-18has not effect on the chondrocytes proliferation, but IL-18can inhibit the upregulation of cell proliferation stimulated by TGF-β.2. IL-18can induce inflammatory response in chondrocyte and synoviocytes, and inhibit the synthesis of aggrecan.3. High level of IL-18in the rats’ knee joints induces the joint degeneration by increasing the inflammation and degrading the cartilage. |
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