独活寄生汤含药血清抑制白细胞介素1β诱导的软骨细胞炎症反应的作用机制研究

Study on mechanism of action of Duhuo Jisheng Tang( 独活寄生汤) medicated serum in inhibiting inflammatory reaction induced by interleukin-1 beta in chondrocytes

目的:探讨独活寄生汤含药血清抑制白细胞介素1β(interleukin-1 beta,IL-1β)诱导的软骨细胞炎症反应的作用机制。方法:将10只2月龄雄性SD大鼠随机分为正常组和独活寄生汤组,独活寄生汤组以9.3 g·kg^(-1)剂量的独活寄生汤灌胃,正常组给予等量生理盐水灌胃;每日灌胃2次,连续1周;末次灌胃后,经腹主动脉取血,分别制备独活寄生汤含药血清及空白血清,低温保存备用。截取6只4周龄SD大鼠膝关节软骨,采用酶消化法分离并培养软骨细胞,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。取生长良好的第2代软骨细胞,采用MTT法检测含药血清培养24 h、36 h、48 h、72 h后的软骨细胞活性,采用酶联免疫吸附法测定不同浓度IL-1β干预后软骨细胞的基质金属蛋白酶(matrix metalloproteinase,MMP)-13含量。将培养好的第2代软骨细胞随机分为空白血清组、模型组、独活寄生汤含药血清组,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;模型组加入浓度为15 ng·m L^(-1)的IL-1β干预24 h后,采用含10%空白血清的DMEM培养;独活寄生汤含药血清组加入浓度为15 ng·m L^(-1)的IL-1β干预24 h后,采用含10%独活寄生汤含药血清的DMEM培养;3组均连续培养48 h,采用Western blot法检测软骨细胞中G蛋白偶联信号传导系统关键调控因子的蛋白表达情况。结果:(1)软骨细胞形态及免疫组化鉴定结果。第2代软骨细胞胞浆丰富,细胞周围可见具有折光性的细胞外基质,细胞长势呈"铺路石"状,胞浆呈棕黄色阳性染色。(2)独活寄生汤含药血清培养不同时间后的软骨细胞活性。含药血清培养24 h、36 h、48 h、72 h后的软骨细胞活性比较,差异有统计学意义(1.01±0.01,1.03±0.02,1.07±0.01,1.02±0.02,F=8.300,P=0.008)。含药血清培养24 h时的软骨细胞活性低于培养48 h时的软骨细胞活性(t=-4.648,P=0.002);与培养36 h、72 h时的软骨细胞活性比较,差异无统计学意义(t=-1.549,P=0.160;t=-0.775,P=0.461)。(3)不同浓度IL-1β干预后的MMP-13含量。在含IL-1β浓度为0 ng·m L^(-1)、5 ng·m L^(-1)、10 ng·m L^(-1)、15 ng·m L^(-1)、20 ng·m L^(-1)、25 ng·m L^(-1)的DMEM中培养24 h后软骨细胞MMP-13含量比较,差异有统计学意义[(0.07±0.01)ng·m L^(-1),(0.08±0.01)ng·m L^(-1),(0.09±0.01)ng·m L^(-1),(0.10±0.01)ng·m L^(-1),(0.08±0.01)ng·m L^(-1),(0.06±0.01)ng·m L^(-1),F=6.823,P=0.001]。未经IL-1β干预时的软骨细胞MMP-13含量与浓度为5 ng·m L^(-1)、20 ng·m L^(-1)、25 ng·m L^(-1)的IL-1β干预后软骨细胞MMP-13含量比较,差异均无统计学意义(LSD-t=-2.049,P=0.055;LSD-t=-2.083,P=0.052;LSD-t=0.496,P=0.626);浓度为10 ng·m L^(-1)、15 ng·m L^(-1)的IL-1β干预后软骨细胞MMP-13含量高于未经IL-1β干预时的软骨细胞MMP-13含量(LSD-t=-3.412,P=0.003;LSD-t=-4.216,P=0.001);浓度为10 ng·m L^(-1)的IL-1β干预后软骨细胞MMP-13含量与浓度为15 ng·m L^(-1)的IL-1β干预后软骨细胞MMP-13含量比较,差异无统计学意义(LSD-t=-0.804,P=0.432)。(4)软骨细胞中G蛋白偶联信号传导系统关键调控因子的蛋白表达。空白血清组、模型组和独活寄生汤含药血清组的Gαs、Gαq、Gαo和Gαi蛋白表达量比较,组间差异均有统计学意义[(0.81±0.09)k Da,(0.31±0.07)k Da,(0.78±0.13)k Da,F=23.669,P=0.001;(0.22±0.04)k Da,(0.14±0.02)k Da,(0.20±0.02)k Da,F=6.500,P=0.031;(0.25±0.02)k Da,(0.12±0.01)k Da,(0.18±0.03)k Da,F=27.214,P=0.001;(0.21±0.02)k Da,(0.26±0.02)k Da,(0.19±0.03)k Da,F=6.882,P=0.028];空白血清组Gαs、Gαq、Gαo蛋白表达量高于模型组(LSD-t=6.134,P=0.001;LSD-t=7.370,P=0.000;LSD-t=3.465,P=0.013),Gαi蛋白表达量低于模型组(LSD-t=2.572,P=0.042);模型组Gαs、Gαq、Gαo蛋白表达量低于独活寄生汤含药血清组(LSD-t=5.766,P=0.001;LSD-t=3.401,P=0.014;LSD-t=2.599,P=0.041),Gαi蛋白表达量高于独活寄生汤含药血清组(LSD-t=3.609,P=0.011)。结论:独活寄生汤含药血清可以抑制IL-1β诱导的软骨细胞炎症反应,其作用机制可能与G蛋白偶联信号传导系统的调控有关,但其具体作用靶点有待进一步深入研究。

Objective ：To explore the mechanism of action of Duhuo Jisheng Tang（独活寄生汤, DHJST） medicated serum in inhibiting inflammatory reaction induced by interleukin - 1 beta（ IL - 1β） in chondrocytes. Methods：Ten 2 - month - old male SD rats were randomly divided into normal group and DHJST group. The rats in DHJST group were intragastric administrated with DHJST in dosage of 9.3 g/kg, while the others in normal group were intragastric administrated with the same dose of normal saline, twice per day for 1 consecutive week. After the last intragastric administration, their blood were fetched out from abdominal aorta and were made into DHJST medicated serum and blank serum respectively and the serum were reserved at low temperature for future use. The knee articular cartilage of six 4 - week - old SD rats were fetched out and the chondrocytes were isolated and cultured by using enzymatic digestion method. The chondrocytes morphology were observed under optical microscope, and immunohistochemical identification were carried out by using type II collagenase. The well - growned second - generation chondrocytes were fetched out, and their cytoactive were detected by using MTr method after cultured in medi- cated serum for 24,36,48 and 72 hours, and the matrix metatloproteinase（MMP） - β content of chondrocytes were detected by using en- zyme -linked immunoadsordent assay（ELISA） after intervention with different concentrations of IL - 1β. The second - generation chondro- cytes were randomly divided into blank serum group, model group and DHJST medicated serum group. The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium（DMEM） supplemented with 10% blank serum, while the chondrocytes in model group and DHJST medicated serum group were intervened by IL - 1β with concentration of 15 ng/ml for 24 hours and then were cultured in I）MEM supplemented with 10% blank serum and 10% DHJST medicated serum respectively. The protein expression of key regulating factor of G protein coupled signal transduction systems in chondrocytes were detected by using Western blot method after the chondrocytes were cultured for continuous 48 hours in the 3 groups. Results ： The second - generation chondrocytes had plentiful kytoplasm and the refractive extracellular matrix（ECM） could be found around the cells. The cells grew in the form of paving - stone and their cytoplasms presented with tawny positive staining. There was statistical difference in the cytoactive between chondrocytes cultured in medicated serum for 24,36,48 and 72 hours （ 1.01 ＋/- 0.01,1.03 ＋/- 0.02,1.07 ＋/- 0.01,1.02 ＋/- 0.02, F = 8. 300, P = 0.008）. The chondrocyte activities were low- er at 24 hours compared to at 48 hours after cultivation in medicated serum（ t = - 4. 648 ,P = 0. 002）. There was no statistical difference in the chondrocyte activities between 24 - hour cultivation and 36 - and 72 - hour cultivation （t = - 1. 549, P = 0. 160;t = -0.775, P = 0. 461 ）. There was statistical difference in the MMP - β contents between chondrocytes cultured in DMEM supplemented with IL - 1β with concentration of 0,5,10,15,20 and 25 ng/ml for 24 hours （ 0.07 ＋/- 0.01,0.08 ＋/- 0.01,0.09 ＋/- 0.01,0. 10 ＋/- 0.01,0.08 ＋/- 0.01,0.06 ＋/-0.01, F = 6. 823 ,P = 0. 001 ）. There was no statistical difference in the MMP - β contents of chondrocytes between pre - intervention by IL- 1β and post- intervention by IL- 1β with concentration of 5,20 and 25 ng/ml respectively（ LSD -t = -2. 049 ,P = 0. 055 ; LSD - t = - 2. 083,P = 0.052 ; LSD - t = 0. 496, P = 0. 626. The MMP - β contents of chondrocytes were higher after intervention by IL - 1βwith concentration of 10 and 15 ng/ml compared to pre - intervention by IL - 1β （ LSD - t = - 3. 412, P = 0. 003 ; LSD - t = -4,216 ,P = 0. 001 ）. There was no statistical difference in the MMP- β contents between chondrocytes intelwented by IL- 1 β with con- centration of 10 ng/ml and those intervented by IL - 1βwith concentration of 15 ng/ml（ LSD - t = - 0. 804 ,P = 0. 432 ）. There was statisti- cal difference in the protein expression of key regulating factor of G protein coupled signal transduction systems including Gαs, Gαq, Gαo and God in chondrocytes between the 3 groups （0.81 ＋/- 0.09,0.31 ＋/- 0.07,0.78 ＋/- 0.β kDa, F = 23. 669, P = 0. 001 ; 0.22 ＋/- 0.04,0.14 ＋/-0.02,0.20 ＋/-0.02 kDa,F =6. 500,P =0. 031 ;0.25 ＋/-0.02,0.12 ＋/-0.01,0.18 ＋/-0.03 kDa,F =27.214,P = 0. 001 ;0.21 ＋/- 0.02,0.26 ＋/- 0.02,0.19 ＋/- 0.03 kDa, F = 6. 882, P = 0. 028 ）. The protein expressions of Gas, Gctq and Gαo were higher and the protein expressions of Gαi were lower in blank serum group compared to model group（LSD - t =6. β4,P =0. 001 ;LSD - t = 7. 370, P = 0. 000 ; LSD - t = 3. 465,P = 0. 0β, LSD - t = 2. 572, P = 0.042 ）. The protein expressions of Gαs, Gαq and Gαo were lower and the protein expressions of Gcti were higher in model group compared to DHJST medicated serum group （ LSD - t -- 5. 766, P = 0. 001 ; LSI） - t = 3. 401 ,P = 0. 014 ;LSD - t - 2. 599,P = 0. 041 ; LSD - t = 3. 609, P = 0.011 ）. Conclusion： DHJST medicated serum can inhibit inflammatory reaction induced by interleukin - 1 beta in chondrocytes. The mechanisms of action may be related to the regulation of G pro- tein coupled signal transduction systems. However,its specific action targets need to be further studied.