| Objectives By means of SD rats in vivo and in vitro experiments, this paper investigates the expression changes of GPR48 at articular cartilage in a post-traumatic osteoarthritis process, and whether there is a relationship of expression of joint control between GPR48 and the inflammatory immune response factor, attempts to further reveal intrinsic the molecular mechanism in the post-traumatic osteoarthritis process, provides a new idea to the research and treatment of post traumatic osteoarthritis.Methods(A) In vitro experiment: 21 SPF female SD rats aged 4 weeks that are killed by cervical dislocation. Medical alcohol soaks 30 min, then take cartilage in the strict aseptic conditions, shear tissues by ophthalmic scissors to do primary culture and sub culture, identify the cartilage cells with toluidine blue staining method and COL-II immunocytochemical staining method. Take the third generation cells, and randomly divide them into group with complete medium(control group), group LPS( 20mg/m L) and group IL-1β( 10ng/m L);extract cellular proteins from three groups each after 0.5h, 1h, 6h, 24 h, 48 h time point, to detect the protein content by BCA method and the expression of GPR48, TLR4 protein by Western Blot method, Image J semi quantitative analysis protein expression.(B) In vivo experiment: 48 SPF female SD rats aged 4 weeks that are randomly divided into control group and experimental group, 12 rats in the control group, and the other 36 in the experimental group. The rats of the experimental group underwent bilateral transection of anterior cruciate ligament(ACL) and medial meniscectomy induced PTOA. The rats in the control group are only opened knee-joint capsule(sham operation), two groups of rats at 2, 4, 6 weeks respectively are sacrificed and took the knee-joint samples fixed in 4% paraformaldehyde for 2 weeks, EDTA decalcified for 2 months, paraffin embedded sections, each group after general staining(HE) and special staining(Masson, VG), to detect the protein expression of articular cartilage GPR48, COL-II, TLR4 by immunohistochemical staining method and observe under optical microscope then grade by IRS score criteria. The data are statistically processed by SPSS17.0 statistical software, and the experimental data are expressed as mean ± standard deviation( x ±s), using the variance analysis test method of single factor and Pearson correlation analysis, P<0.05 said the difference is statistically significant.Results(A) In vitro: 1 Toluidine blue staining: the normal cartilage cells secrete characteristic acidic glycosaminoglycans that can be stained by toluidine blue with purple red, and cartilage cells stained by toluidine blue presents metachromatic.2 COL-II immunocytochemistry staining: CO L-II is the chondrocyte-specific secretions, so using COL-II immunocytochemistry to prove chondrocytes COL-II presents positive, brown particles appears in cytoplasm. 3 Western Blot method to detect the expression of GPR48 and TLR4: GPR48 in the experimental group(LPS and IL-1β), the expression decreases in prolonged time(P<0.05); in the control group, the difference change between the expression and time is little(P>0.05); TLR4 in the experimental group(LPS and IL-1β), the expression increases(P<0.05) in prolonged time, in the control group, the difference change between the expression and time is little(P>0.05).In the intervention group(LPS and IL-1β), The expression quantity between GPR48 and TLR4 is negative correlation at different time points in the IL-1β group(r = 0.991,P<0.05); The expression quantity between GPR48 and TLR4 is negative correlation at different time points in the LPS group(r = 0.997, P<0.05).(B) In vivo: successfully prepared SD rat animal model of osteoarthritis.1 HE staining: control group, the surface of articular cartilage is smooth, chondrocytes of cartilage lacuna arrange regularly in neat rows, the subchondral bone trabecula is thick and integrated. Experimental group, after two weeks articular surface is relatively good, chondrocytes of cartilage lacuna are neatly arranged, and clusters of cells are visible, the subchondral bone trabecular are arranged relatively better, Bone trabecular thickness increases; experimental group after four weeks cartilage surface cells are lost, the cell clones appears that is relatively normal cell morphology, the more disordered arrangement, the subchondral bone trabecular increases, trabecular gap become small. After six weeks in the experimental group cartilage cells lose severely, cell morphology is abnormal and the arrangement is disorder, tide line forward, the subchondral bone trabecula increases, trabecular gap become smaller, and disordered arrangement. 2 Articular cartilage Masson trichrome: the result shows that collagen fibers of bone and cartilage matrix are dyed blue, erythrocyte and muscle fibers red, and nucleus blueblack. In experimental group, the cartilage matrix reduces with time, matrix staining lightens, red stained area reduces. No significant change in the control group. And collagen content per unit area of the experimental group and control group notably decrease after two weeks, four weeks, six weeks, and the difference is statistically significant(P<0.05). 3 VG staining: the result shows that collagen fibers are dyed red. In experimental group, the cartilage matrix decreases with time, matrix stain lightens, red stained area reduces. No significant change in the control group. And collagen content per unit area of the experimental group and control group notably decrease after two weeks, four weeks, six weeks, and the difference is statistically significant(P<0.05). 4 Immunohistochemical staining method detecting the protein expression of articular cartilage GPR48, COL-II and TLR4: The positive staining of COL-II protein presents brownish yellow granules, both expressed in chondrocytes. GPR48 and COL-II protein expression in the experimental group at each time point are both lower than that of control group(P<0.05). The positive staining of TLR4 protein presents brownish yellow granules, located membrane and cytoplasm, mainly expressed in chondrocytes, osteoblasts and bone cells also seen little expression. TLR4 protein expression in the experimental group at each time point are both higher than that of control group(P<0.05), and the expression between GPR48 and TLR4 is negative correlation at different time points in experimental group(r =-0.925,P<0.05).Conclusions 1 This experiment successfully replicates the SD rat model of osteoarthritis, and to certain extent reflects the pathological changes of osteoarthritis, and establishes a good platform for the later study. 2 The experiment in vitro shows that, articular chondrocytes are stimulated respectively by inflammatory cytokines IL-1β, LPS, the expression of GPR48 is down regulation as a result of stimulation, while TLR4 up, the expresson quatity between the two is negative correlation, showed that in inflammatory conditions, GPR48 negatively regulates chondrocyte function in inflamamatory conditions, GPR48 negatively regulates the chondrocyte immune inflammatory response. 3 The experiment in vivo shows that, with the post-traumatic osteoarthritis progression, the expression of arthrodial cartilage GPR48 gradually decreases, while TLR4 gradually increases, which illustrates GPR48 plays an important role in the process of osteoarthritis regulation. 4 In the developing process of post-traumatic osteoarthritis, there is a negative correlation between GPR48 and inflammation response factor TLR4 expression, which indicates GPR48 and its inflammatory reaction both participate in the regulation of osteoarthritis, thus it provides a new idea to understand the molecular mechanisms of osteoarthritis inflammation and establish new therapeutic target. |
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