跳骨片含药血清抑制脂多糖诱导的软骨细胞炎症反应的作用机制研究

Study on mechanism of action of Tiaogu Pian(跳骨片) medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes

目的:探讨跳骨片含药血清抑制脂多糖诱导的软骨细胞炎症反应的作用机制。方法:将10只8周龄雄性SD大鼠随机分为跳骨片组和空白组,跳骨片组以0.32 g·kg-1剂量的跳骨片灌胃,空白组给予等量生理盐水灌胃;每天灌胃1次,连续7 d;末次灌胃1 h后,经腹主动脉取血,分别制备跳骨片含药血清和空白血清,低温保存备用。从10只4周龄SD大鼠膝关节软骨中分离软骨细胞并培养,光学显微镜下观察软骨细胞形态,并用Ⅱ型胶原酶免疫组化鉴定。将培养好的第2代软骨细胞随机分为空白血清组、模型组、跳骨片含药血清组,其中空白血清组以含10%空白血清的培养基(dulbecco modified eagle medium,DMEM)培养;模型组以浓度为10 ng·mL-1的脂多糖和含10%空白血清的DMEM培养;跳骨片含药血清组以浓度为10 ng·mL-1的脂多糖和含10%跳骨片含药血清的DMEM培养;3组均连续干预培养8 h,采用酶联免疫吸附法检测软骨细胞基质金属蛋白酶(matrix metalloproteinase,MMP)3、MMP9含量,采用荧光定量RT-PCR法检测软骨细胞中Wnt/β-catenin信号通路相关基因表达水平,采用Western blot法检测软骨细胞中β-链蛋白(β-catenin)、卷曲蛋白-2(Frizzled-2)的蛋白表达量,采用免疫荧光检测法检测软骨细胞中β-catenin、糖原合成酶激酶-3β(glycogen synthasc kinase-3β,GSK-3β)、蛋白多糖1(proteoglycans 1,PGS1)的蛋白表达量。结果:(1)软骨细胞免疫组化鉴定结果。第2代软骨细胞胞浆及细胞膜呈棕黄色阳性染色,具有典型的软骨细胞生物学特征。(2)软骨细胞MMP3、MMP9含量。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组的软骨细胞MMP3、MMP9含量比较,组间差异均有统计学意义[(34.019±1.036)ng·mL-1,(44.645±2.473)ng·mL-1,(32.941±1.792)ng·mL-1,F=36.060,P=0.000;(1.348±0.038)ng·mL-1,(1.562±0.112)ng·mL-1,(1.331±0.015)ng·mL-1,F=11.319,P=0.000];模型组软骨细胞MMP3、MMP9含量均高于空白血清组(LSD-t=-7.016,P=0.000;LSD-t=-3.768,P=0.003);跳骨片含药血清组软骨细胞MMP3、MMP9含量均低于模型组(LSD-t=7.652,P=0.000;LSD-t=4.066,P=0.002);空白血清组软骨细胞MMP3、MMP9含量与跳骨片含药血清组比较,差异均无统计意义(LSD-t=0.635,P=0.549;LSD-t=0.299,P=0.770)。(3)软骨细胞中Wnt/β-catenin信号通路相关基因的表达。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、GSK-3β、Frizzled-2、Wnt-4、CKI-ε基因表达量比较,组间差异均有统计学意义(1.000±0.275,2.258±0.206,1.431±0.304,F=36.709,P=0.000;1.000±0.133,0.417±0.104,0.842±0.094,F=29.259,P=0.000;1.000±0.191,1.737±0.238,1.445±0.337,F=7.027,P=0.015;1.000±0.341,3.801±0.579,1.876±0.388,F=71.903,P=0.000;1.000±0.309,2.208±0.708,1.441±0.421,F=64.178,P=0.000);模型组软骨细胞中β-catenin、Frizzled-2、Wnt-4、CKI-ε基因表达量均高于空白血清组(LSD-t=-8.431,P=0.000;LSD-t=-3.723,P=0.005;LSD-t=8.062,P=0.000;LSD-t=-11.235,P=0.000),GSK-3β基因表达量低于空白血清组(LSD-t=7.397,P=0.000);跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2、Wnt-4、CKI-ε基因表达量均低于模型组(LSD-t=5.541,P=0.000;LSD-t=1.477,P=0.017;LSD-t=8.062,P=0.000;LSD-t=6.882,P=0.000),GSK-3β基因表达量高于模型组(LSD-t=-5.387,P=0.000);空白血清组软骨细胞中β-catenin、Wnt-4、CKI-ε基因表达量均低于跳骨片含药血清组(LSD-t=-2.289,P=0.018;LSD-t=-3.658,P=0.005;LSD-t=-4.352,P=0.002);空白血清组软骨细胞中GSK-3β、Frizzled-2基因表达量与跳骨片含药血清组比较,差异均无统计学意义(LSD-t=2.009,P=0.075;LSD-t=-3.658,P=0.051)。(4)软骨细胞中β-catenin、Frizzled-2的蛋白表达。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2蛋白表达量比较,组间差异均有统计学意义(0.449±0.063,0.746±0.156,0.549±0.056,F=5.323,P=0.026;1.348±0.038,1.562±0.112,1.331±0.015,F=6.291,P=0.034);模型组软骨细胞中β-catenin、Frizzled-2蛋白表达量高于空白血清组(LSD-t=-11.235,P=0.005;LSD-t=-3.104,P=0.021);跳骨片含药血清组软骨细胞中β-catenin、Frizzled-2蛋白表达量低于模型组(LSD-t=6.883,P=0.037;LSD-t=3.039,P=0.023);空白血清组软骨细胞中β-catenin蛋白表达量低于跳骨片含药血清组(LSD-t=-4.352,P=0.002);空白血清组软骨细胞中Frizzled-2蛋白表达量与跳骨片含药血清组比较,差异无统计学意义(LSD-t=-0.065,P=0.950)。(5)软骨细胞中β-catenin、GSK-3β、PGS1的蛋白表达。脂多糖干预8 h后,软骨细胞中β-catenin、GSK-3β、PGS1蛋白染色明显,呈绿色;空白血清组、模型组和跳骨片含药血清组软骨细胞中β-catenin、GSK-3β、PGS1蛋白表达量比较,组间差异均有统计学意义(0.014±0.002,0.029±0.006,0.018±0.002,F=9.910,P=0.013;0.380±0.011,0.237±0.015,0.287±0.002,F=56.639,P=0.000;0.034±0.003,0.022±0.002,0.029±0.003,F=27.232,P=0.001);模型组软骨细胞β-catenin蛋白表达量高于空白血清组(LSD-t=-4.103,P=0.006),GSK-3β、PGS1蛋白表达量低于空白血清组(LSD-t=1.048,P=0.000;t=7.365,P=0.000);跳骨片含药血清组软骨细胞β-catenin蛋白表达量低于模型组(LSD-t=-3.548,P=0.012),GSK-3β、PGS1蛋白表达量高于模型组(LSD-t=-3.657,P=0.011;LSD-t=-3.273,P=0.017);空白血清组软骨细胞中β-catenin蛋白表达量与跳骨片含药血清组比较,差异无统计学意义(LSD-t=-0.554,P=0.599);空白血清组软骨细胞中GSK-3β、PGS1蛋白表达量高于跳骨片含药血清组(LSD-t=6.827,P=0.000;LSD-t=4.092,P=0.010)。结论:跳骨片含药血清可以抑制脂多糖诱导的软骨细胞炎症反应,延缓关节软骨退变。其作用机制可能与Wnt/β-catenin信号通路的调控有关,其中β-catenin、Frizzled-2、GSK-3β、Wnt-4、CKI-ε基因可能是该信号通路的重要靶点。但是由于引起OA的因素众多且跳骨片含药血清成分多且复杂,有待于进一步研究证实。

Objective：To explore the mechanism of action of Tiaogu Pian（ 跳骨片, TGP）medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes. Methods： Ten 8 - week - old male SD rats were randomly divided into TGP group and blank group. The rats in TGP group were intragastric administrated with TGP in dosage of 0.32 g/kg, while the others in blank group were intragastric administrated with the same dose of normal saline, once per day for 7 consecutive days. At 1 hour after the last intragastric administration, their blood were fetched out from abdominal aorta and were made into TGP medicated serum and blank serum respectively and the serum were reserved at low temperature for future use. The chondrocytes of ten 4 - week - old SD rats were isolated from knee artic- ular cartilage and were cultured. The chondrocytes morphology were observed under optical microscope, and immunohistochemical identifica- tion were carried out by using type 11 collagenase. The second - generation chondrocytes were randomly divided into blank serum group, mod- el group and TGP medicated serum group. The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium （DMEM） supplemented with 10% blank serum. The chondrocytes in model group were cultured in DMEM supplemented with lipopolysac- charide（ LPS）with concentration of 10 ng/ml and 10% blank serum. The chondrocytes in TGP medicated serum group were cultured in DMEM supplemented with LPS with concentration of 10 ng/ml and 10% TGP medicated serum. The chondrocytes in the 3 groups were in- tervened and cultured for continuous 8 hours. The content of matrix metalloproteinase（MMP） 3 and MMP9 in chondrocytes were detected by using enzyme- linked immunoadsordent assay（ ELISA ）. The expression levels of gene related to Wnt/[3- catenin signaling pathway in chondrocytes were detected by using fluorescence quantitative RT - PCR method. The protein expressions of β - catenin and Frizzled - 2 in chondrocytes were detected by using Western blot method. The protein expressions of β - catenin, glycogen synthasc kinase -3β （GSK - 3β） and proteoglycans 1 （PGS1）in chondrocytes were detected by using immunofluorescence assay（IFA）. Results：The second- generation chondrocytes had typical biological characteristics of chondrocytes and their endochylemas and cytomembranes presented with brown - yellow positive staining. After 8 - hour intervention by LPS, there was statistical difference in the content of MMP3 and MMP9 in chondro- cytes between blank serum group, model group and TGP medicated serum group （ 34. 019 ＋/- 1. 036,44. 645 ＋/- 2. 473,32. 941 ＋/- 1. 792 ng/ml,F = 36. 060,P =0.000; 1. 348 ＋/-0.038,1. 562 ＋/-0. 112,1. 331 ＋/-0.015 ng/ml,F = 11. 319 ,P =0.000）. The content of MMP3 and MMP9 in chondrocytes were higher in model group compared to blank serum group（ LSD -t = -7. 016 ,P = 0. 000 ;LSD -t = -3. 768 ,P =0.003 ）and were lower in TGP medicated serum group compared to model group（ LSD -t = 7. 652 ,P = 0. 000; LSD -t = 4. 066, P = 0. 002）. There was no statistical difference in the content of MMP3 and MMP9 in chondrocytes between blank serum group and TGP medicated serum group （ LSD - t = 0. 635, P = 0.549 ; LSD - t = 0. 299,P = 0. 770）. After 8 - hour intervention by LPS, there was sta- tistical difference in gene expression levels of β- catenin, GSK- 3β, Frizzled- 2, Wnt- 4 and CKI-ε between blank serum group, model group and TGP medicated serum group （ 1. 000 ＋/- 0. 275,2. 258 ＋/- 0. 206,1.431 ＋/- 0. 304, F = 36. 709, P = 0. 000 ; 1. 000 ＋/- 0.β3, 0.417 ＋/- 0.104,0. 842 ＋/- 0. 094, F = 29. 259, P = 0. 000 ; 1. 000 ＋/- 0.191,1. 737 ＋/- 0. 238,1. 445 ＋/- 0. 337, F = 7. 027, P = 0.015 ; 1. 000 ＋/- 0. 341,3. 801 ＋/- 0. 579,1. 876 ＋/- 0. 388, F = 71. 903, P = 0. 000 ; 1. 000 ＋/- 0. 309,2. 208 ＋/- 0. 708,1.441 ＋/- 0.421, F = 64. 178, P = 0. 000）. The gene expression levels of β - catenin, Frizzled -2, Wnt- 4 and CKI - ein chondrocytes were higher and the gene expression levels of GSK - 3βwere lower in model group compared to blank serum group （ LSD - t = - 8. 431, P = 0. 000 ; LSD - t = - 3. 723, P = 0. 005 ; LSD - t = 8. 062,P = 0. 000 ; LSD - t = - 11. 235, P = 0. 000, LSD - t = 7. 397, P = 0. 000 ）. The gene expression lev- els of β - catenin, Frizzled - 2, Wnt - 4 and CKI - ε in chondroeytes were lower and the gene expression levels of GSK - 3β were higher in TGP medicated serum group compared to model group（ LSD - t = 5. 541, P = 0. 000 ; LSD - t = 1. 477, P = 0. 017 ; LSD - t = 8. 062, P = 0. 000 ; LSD - t = 6. 882,P = 0. 000 ; LSD - t = - 5. 387, P = 0. 000）. The gene expression levels of β - catenin, Wnt - 4 and CKI - 8 in chondroeytes were lower in blank serum group compared to TGP medicated serum group （ LSD - t = - 2. 289, P = 0. 018; LSD - t = - 3. 658,P = 0. 005 ; LSD - t = - 4. 352, P = 0. 002）. There was no statistical difference in gene expression levels of GSK - 3β and Frizzled - 2 between blank serum group and TGP medicated serum group （ LSD - t = 2. 009, P = 0. 075 ; LSD - t = - 3. 658,P = 0.051 ）. Af- ter 8 - hour intervention by LPS, there was statistical difference in protein expressions of β - catenin and Frizzled - 2 between blank serum group, model group and TGP medicated serum group （0. 449 ＋/-0. 063,0. 746 ＋/-0. 156,0. 549 ＋/-0. 056, F = 5. 323, P = 0. 026; 1.348 ＋/-0.038;1.562＋/-0. 112;1.331 ＋/-0. 015,F=6.291,P=0. 034）.The protein expressions of β - catenin and Frizzled -2 in ehondrocytes were higher in model group compared to blank serum group （ LSD - t = - 11. 235, P = 0. 005 ; LSD - t = - 3. 104, P = 0. 021 ）. The protein expressions of β - catenin and Frizzled - 2 in ehondrocytes were lower in TGP medicated serum group compared to model group（ LSD - t = 6. 883,P = 0. 037 ; LSD - t = 3. 039, P = 0. 023 ）. The protein expressions of β - catenin in chondrocytes were lower in blank serum group compared to TGP medicated serum group（ LSD -t = -4. 352, P = 0. 002 ）. There was no statistical difference in pro- tein expressions of Frizzled - 2 between blank serum group and TGP medicated serum group （ LSD - t = - 0. 065, P = 0. 950 ）. After 8 - hour intervention by LPS,the chondrocytes presented with obvious green staining of β - catenin, GSK -3β and PGS1 protein. There was sta- tistical difference in protein expressions of β -catenin, GSK- 3β and PGSI between blank serum group, model group and TGP medicated serum group（0. 014 ＋/- 0. 002,0. 029 ＋/- 0. 006,0. 018 ＋/- 0. 002, F = 9. 910, P = 0. 0β ; 0. 380 ＋/- 0. 011,0. 237 ＋/- 0. 015, 0. 287 ＋/- 0. 002, F = 56. 639, P = 0. 000 ;0.034 ＋/- 0. 003,0. 022 ＋/- 0.002,0. 029 ＋/- 0. 003, F = 27. 232, P = 0.001 ）. The protein ex- pressions of β - catenin were higher and the protein expressions of GSK -3β and PGS1 were lower in model group compared to blank serum group （ LSD - t = - 4.103, P = 0. 006 ; LSD - t = 1. 048, P = 0. 000 ; t = 7. 365, P = 0. 000）. The protein expressions of β - catenin were low- er and the protein expressions of GSK -3β and PGS1 were higher in TGP medicated serum group compared to model group（ LSD - t = - 3. 548,P = 0. 012 ; LSD - t = - 3. 657,P = 0.011 ; LSD - t = - 3. 273, P = 0. 017 ）. There was no statistical difference in protein expres- sions of β - catenin between blank serum group and TGP medicated serum group（ LSD - t = -0. 554 ,P = 0.599 ）. The protein expressions of GSK - 3βand PGS1 were higher in blank serum group compared to TGP medicated serum group （ LSD - t = 6. 827, P = 0. 000 ; LSD - t = 4. 092 ,P = 0.010）. Conelusion：TGP medicated serum can inhibit inflammatory reaction induced by LPS in chondrocytes and delay the ar- ticular cartilage degeneration. The mechanisms of action may be related to the regulation of Wnt/β - catenin signaling pathway, in which β - catenin gene, Frizzled - 2 gene, GSK - 3β gene, Wnt - 4 gene and CKI - ε gene may be the important action targets. However, many factors can cause OA and there are many complicated ingredients in TGP medicated serum ,further studies are needed to confirm the specific action targets.