The Influence Of LPS-induced Expression Of COX-2 And PPAR-γ On The Differentiation Of Orbital Preadipocytes In Thyroid-associated Ophthalmopathy

Objective:To investigate the effects of lipopolysaccharide (LPS)-induced inflammation on the cyclooxygenase-2 (COX-2) expression and prostaglandin E2(PGE2) secretion of normal-derived and thyroid-associated ophthalmopathy (TAO)-derived orbital fibroblasts.Methods:Orbital adipose tissues were obtained from patients with eyeball atrophy or TAO patients undergoing surgery.The orbital fibroblasts cultured from orbital adipose tissues were divided into Group A (normal control) and B (TAO). Both groups were subdivide into Group A0-A7 and Group B0-B7 respectively. The orbital fibroblasts in Group Ao and Bo were incubated in culture medium without any intervention. Fibroblasts in Group A1-A3 and Group B1-B3 were incubated in culture medium containing various concentration of LPS (10 ng/ml,100 ng/ml, 1000 ng/ml) for 24 hours respectively, while fibroblasts in Group A4-A7 and B4-B7 were incubated in culture medium containing 1000 ng/ml LPS for various stimulating time(4h,8h,12h,24h). The expression of COX-2 mRNA and protein of all groups were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western-blot, and the level of PGE2 in the supernatant were detected by enzyme linked immunosorbent assay (Elisa).Results:There were no significant differences in the expression of COX-2 mRNA and protein and the level of PGE2 between Group A0 and Bo (P>0.05). COX-2 mRNA and protein expression and PGE2 level were significantly enhanced in Group B compared with those in Group A when they were treated with the same concentration and stimulating time of LPS(P<0.05).When they were treated with various concentration of LPS(10 ng/ml,100 ng/ml, 1000ng/ml) for 24 hours, the expression of COX-2 mRNA and protein of orbital fibroblasts in Group A2 and A3 were significantly stronger than those in Group A0 and A1 (P<0.05).However, there was no significant difference between Group A2 and A3, or Group A0 and A1(P>0.05). Group A1, A2 and A3 had enhanced concentration of PGE2 in the supernatant compared with Group A0 (P<0.05), among them, Group A3 had the highest PGE2 level. Group B1, B2 and B3 had significantly enhanced expression of COX-2 mRNA and protein when compared with Group Bo (P<0.05), among them, Group B2 and B3 had the highest COX-2 mRNA expression, while Group B3 had the highest protein expression. Group B1, B2 and B3 had significantly enhanced concentration of PGE2 in the supernatant compared with Group Bo (P<0.05), among them, Group B2 and B3 had the highest PGE2 level. When they were treated with 1000 ng/ml LPS for different time (4h,8h, 12h,24h), Group A4, A5, A6 and A7 had significantly enhanced expression of COX-2 mRNA and protein when compared with Group A0 (P<0.05). However, there were no significant differences in any two groups among these four groups(P>0.05). Group A4, A5, A6 and A7 had markedly increased level of PGE2 compared with Group A0 (P<0.05), among them, Group A4 had the highest concentration of PGE2 in the supernatant. Group B4,B5,B6 and B7 had markedly increased expression of COX-2 mRNA and protein and level of PGE2 compared with Group Bo (P<0.05), among them, Group B5 had the highest expression of COX-2 mRNA and Group B4 had the highest concentration of PGE2 in the supernatant.As for the expression of COX-2 protein, there were no significant differences in any two groups among GroupB4-B7 (P>0.05).Conculsion:LPS can induce inflammation in normal and TAO derived orbital fibroblasts and increase the expression of COX-2 and PGE2. TAO-derived orbital fibroblasts had significantly enhanced expression of COX-2 mRNA and protein and secretion of PGE2 than normal-derived cells when they were treated with the same concentration and stimulating time of LPS. Objective:To investigate the influence of COX-2 and peroxisome proliferator activated receptor-γ(PPAR-γ) expression change caused by LPS-induced inflammation on the differentiaion of TAO-derived orbital preadipocytes and explore the relationship among them.Methods:Orbital adipose tissues were obtained from TAO patients undergoing orbital decompression surgery. The orbital preadipocytes cultured from orbital adipose tissues were divided into Group A and Group B. In Group A, the orbital preadipocytes were incubated in culture medium containing 1000 ng/ml LPS for 8 hours before stimulated to differentiate into mature adipocytes with differentiation mediumⅠandⅡ. No LPS or other intervention measures was used in Group B before induced to differentiate into mature adipocytes with the same differentiation medium as the Group A. Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining. The value of optical absorption was measured at 492 nm with enzyme-linked immunosorbent assay instrument. The expression of COX-2 and PPAR-γmRNA of both groups were detected by RT-PCR. The expression of COX-2 and PPAR-γprotein in both groups were detected by Western-blot and the level of PGE2 in the supernatant were detected by Elisa.Results:Oil red O staining showed no obvious morphological difference between differentiated cells of both group.The light absorption value (A) of differentiated cells of Group A (1.02±0.08)was higher than that of Group B (0.74±0.06) (P<0.05).Expression of PPAR-y mRNA and protein of differentiated cells in both groups enhanced significantly, while the expression of COX-2 mRNA and protein and the level of PGE2 decreased markedly compared with those of their own pre-differentiated cells (P<0.05). Both the pre-differentiated and differentiated cells in Group A had significantly enhanced expression of COX-2 and PPAR-y mRNA and protein and concentration of PGE2 in the supernatant when compared with the pre-differentiated and differentiated cells in Group B (P＜0.05).Conculsion:LPS-induced inflammation can promote the differentiation of TAO-derived orbital preadipocytes.The expression of PPAR-y enhanced significantly while the expression of COX-2 and PGE2 attenuated markedly when the orbital preadipocytes were differtiated into adipocytes.Objective:To investigate the effects of mevastatin on the expression of COX-2 and PPAR-γand differentiation of TAO-derived orbital preadipocytes, and to explore its modulation effects on LPS-induced inflammation and the differentiation of TAO-derived orbital preadipocytes in vitro.Methods:Experimental group:a. The effects of Mevastatin intervention on LPS-induced COX-2 expression and PGE2 secretion by TAO-derived orbital fibroblasts- The orbital fibroblasts cultured from orbital adipose tissues were divided into Group A (LPS group), Group B (LPS combined with Mevastatin group) and Group C (control group). The orbital fibroblasts in Group A were stimulated with 1000 ng/ml LPS for 8 hours, whereas those in group B were treated with LPS combined with 5μM(B1 group),10μM(B2 group) or 20μM (B3 group) mevastatin for 8 hours. The orbital fibroblasts in Group C were cultured routinely without any intervention as control; b.The effects of Mevastatin intervention on the differentiation and PPAR-y expression of TAO-derived orbital preadipocytes-Group A in a. above were subdivided into Group A1-A6.1000 ng/ml LPS had been treated for 8 hours before all orbital preadipocytes were induced to differentiate into adipocytes. All groups were stimulated to differentiate into mature adipocyte with cocktail differentiation medium.The entire course of differentiation was 10 days.The differentiation of orbital preadipocytes in Group A1 were induced with routine inducer, while those in Group A2, A3, and A4 were interfered with 5μM(A2),10μM(A3) or 20μM(A4) mevastatin during the whole process of differentiation. The differentiation of orbital preadipocytes in Group A5 and A6 were interfered with 10μM mevastatin at the 4th day(A5) or 8th day(A6) of differentiation process until the entire course was over. Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining. The value of optical absorption was measured at 492 nm with enzyme-linked immunosorbent assay instrument. Expression of COX-2 and PPAR-y mRNA was detected by RT-PCR. Expression of COX-2 and PPAR-y protein was detected by Western-blot. The level of PGE2 in the supernatant were detected by Elisa.Results:The expression of COX-2 mRNA and protein and the level of PGE2 in Group B decreased markedly compared with those in Group A (P<0.05). With the increasing concentration of Mevastatin,the expression of COX-2 mRNA and protein and the level of PGE2 in Group B1,B2,B3 decreased successively(P<0.05). The expression of COX-2 mRNA and protein and the level of PGE2 in Group C decreased significantly compared with those in Group A, B1 and B2 (P＜0.05), while there were no significant differences between Group C and B3(P>0.05). The light absorption value (A) and PPAR-y mRNA and protein expression of differentiated cells in Group A1-A4 decreased successively, and there were significant differences in any two groups among these groups(P<0.05). The value A and PPAR-γmRNA and protein expression of differentiated cells in Group A1, A5 and A3 decreased successively, and the difference in any two groups among these three groups was significant, however, there were no significant difference in Group A1 and A6.Conclusion:Mevastatin can inhibit LPS-induced COX-2 expression and PGE2 secretion of TAO-derived orbital fibroblasts in vitro. It can also inhibit the differentiation and PPAR-y expression of TAO-derived orbital preadipocyte cultured in vitro. The degree of inhibition is not only concentration dependent but also associated with the stage of differentiation. The more earlier in the differentiation, the stronger the inhibition.