Fas, Caspase-3, Bcl-2 Expression In Osteoarthritis Cartilage And Significance

INTRODUCTIONOsteoarthritis, is not only the most frequent articular disease,but the most common reason for articulur pain of the aged people, which severely affect their life qualities. Over the last ten years, along with the increase of activity and intensity of modern people, the incidence of OA showed a new feature: the age of OA onset is younger and younger. But the methods of clinical treatment to OA includes reducing articular pains, improving articular function and replacement of joint, in short of effective methods to control and treat the key of OA (cartlage damage and osteophyte formation). The whole synovial joint including cartilage, synovium and the bone below cartilage are all involved in OA and each has the relatively independent ability to cause OA. No matter what is the original damage, the final pathway is cartilage damage, joint remodeling and osteosclerosis below cartilage. The machanism of OA is not clear yet, involving the whole and local factors, which is perhaps a kind of integrative machanism. Many research work showed that apoptosis participate in OA. The vacuity in some areas of OA articular cartilage especially in calcification layer was confirmed as the effect of apoptosis. The more apoptotic cells mean the less functional chondrocytes, which lead to the less ECM synthesized by chondrocytes, which was believed as the beginning of OA cartilage damage. So far to now, the machanism of apoptosis in OA is not clear. With the more apoptosis-related factors discovered, the machanism of apoptosis seams more complicated. The three important apoptosis-related factors of them: Fas, caspase-3 and bcl-2 were selected in our study.OBJECTIVETo detect the expression of Fas, caspase-3, bcl-2 protein and mRNA in two zones of OA cartilage showing the least and the most cartilage damage, and to show the functions of these three important apoptosis-related factors in OA cartilage damage. METHODSCartilage samples were collected from 10 OA patients (Female:4, Male: 6; aged 55-68 years), definitely diagnosed by clinical and pathological proof. All were from 12 knees (Left: 5, Right: 7) undergoing surgery knee replacement. Observing the general appearance, samples were taken from each zone showing the least (Min) and most (Max) OA damage, with the size of 1.5×1.0×0.5cm³. Each was vertically subdivided into two, randomly used for making paraffin sections and for RT-PCR. Before making paraffin sections, the samples were fixed in 10% neutral aormaldehyde solution for more than 24 hours and decalcificated in ethanedioic acid fluid for 21 days. H-E staining and Safranin O/Fast Green staining were performed on each specimen. The degree of cartilage damage was evaluated using the modified Mankin grading scheme. The expression of p53, caspase-3, bcl-2 protein in OA Min zones and in Max zones were detected through immunohistochemistry and ratios of positive cells were recorded under the microscope, using PBS in the place of fires-used antibodies as the negative control. The apoptosis in OA Min zones and in Max zones were detected through the terminal-deoxynu-transferase mediated nick end labeling (TUNEL) method, labeled by FITC-dUTP, re-stained by PI, and ratios of positive cells were recorded under the fluorescence microscope, using the solution without TDT in the place as the negative control. The expression of p53, caspase-3, bcl-2 mRNA in OA Min zones and in Max zones were detected through RT-PCR techniques. Fas primer: Left 5'-CTC CAA GGG ATT GGA ATT GA-3', Right 5'-TTG GTG TTG CTG GTG AGT GT-3' (Serial number: NM₀00043, Scope: 440-838, Size: 399 bp). caspase-3 primer: Left 5'-TGG AAT TGA TGC GTG ATG TT-3', Right 5'-GTC GGC ATA CTG TTT CAG CA-3' (Serial number: NM₀04346, Scope: 554-948, Size:395 bp). Bcl-2 primer: Left 5'-GGG TAC GAT AAC CGG GAG AT-3', Right 5'-CTG AAG AGC TCC TCC ACC AC-3' (Serial number: NM₀00633, Scope: 516-910, Size: 395 bp). Intro-compared usingp-actin, p-actin primer: Left 5'-GGC ATC CTC ACC CTG AAG TA-3', Right 5'-GGG GTG TTG AAG GTC TCA AA-3' (Serial number: NM₀01101, Scope: 261-463, Size 203 bp). The product along with marker were performed in agarose gel electrophoresis after split in Trizol, low temperature centrifugalization, reverse transcription and amplification in PCR reaction system (conditions: pre-degeneration 94℃4min, degeneration 94℃30s, renaturation 60℃30s, elongation 72℃lmin, elongation 72℃10min, 30 cycles). Observed and recorded under the ultraviolet camera, the average gray of each electrophoresis band was measured through computer software. The ratio toβ-actin was defined as the relative value of mRNA. All data were analysed through Concise Statistics 10.34 software, with P＜0.05 representing statistical significance.RESULTSThe OA cartilage in Min zones appeared whitish or whitish-yellowish, tarnished, smooth, few slightly fissured. While cartilage in Max zones showed yellowish, rough and uneven, thin, fissured deeply even into the bone below. H-E staining showed chondrocytes were relatively dense and well-distributed, with a few swollen cells in Min zones, while Max zones showed evident hypocellularity, more vacuity, more swollen cells with bigger vacuolus and some cells assembled, Safranin O/Fast Green staining showed that cellular nuclei were stained brownish-black, cytoplasm green, ECM orange yellow and severe reduction of the safranin O staining in OA Max zones compared with Min zones. The degree of cartilage damage evaluated using the modified Mankin grading scheme of OA Max zones (7.08±1.00) was much more than that of Min zones (1.92±1.62) with statistical significance (P＜0.05). Immunohistochemistry showed the expression of Fas, caspase-3, bcl-2 protein in OA Max and Min zones, appeared brownish-yellow, none in the negative controls. Ratios (%) of Fas, caspase-3, bcl-2 positive cells in OA Max zones (26.45±4.67, 28.00±5.46, 24.15±3.57) were more than those in Min zones (20.17±2.63, 20.54±4,24, 20.04±3.94) with statistical significance (P＜0.05), which showed the increase of Fas, caspase-3, bcl-2 protein expression in OA severe-damaged cartilage. TUNEL method showed TUNEL positive cells in OA Max and Min zones with green light under the fluorescence microscope, while TUNEL negative cells and all cells in the negative controls seemed red. Ratios (%) of TUNEL positive cells in OA Max zones (31.36±5.86) were more than those in Min zones (23.05±5.17) with statistical significance (P＜0.05), which showed more apoptosis occurred in OA severe-damaged cartilage. RT-PCR showed the expression of Fas, caspase-3, bcl-2 mRNA in OA Max and Min zones, appeared white even bands in agarose gel electrophoresis under the ultraviolet camera. The relative values of Fas, caspase-3, bcl-2 mRNA in OA Max zones (1.29±0.12, 1.37±0.10, 1.19±0.04) were more than those in Min zones (0.99±0.07, 1.02±0.10, 0.99±0.04) with statistical significance (P＜0.05), which showed the increase of Fas, caspase-3, bcl-2 mRNA expression in OA severe-damaged cartilage, consistent with their protein expression.CONCLUSIONOur study showed that the expression of Fas, caspase-3, bcl-2 protein and mRNA besides apoptotic chondrocytes in OA articular cartilage were coincident with the level of OA cartilage damage, which showed apoptosis and apoptosis-related factors participated in OA cartilage damage. This might be a machanism of OA onset, so proper regulations towards apoptosis and apoptosis-related factors might be an effective way to treat OA cartilage damage.