四妙散调节软骨细胞凋亡与自噬治疗膝骨关节炎大鼠的实验研究
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摘要
目的:通过膝骨关节炎大鼠模型研究四妙散调节软骨细胞凋亡与自噬治疗膝骨关节炎(KOA)大鼠的作用机制。方法:8周龄SD大鼠27只,随机分为正常对照组、KOA模型组、四妙散组,KOA模型组和四妙散组采用前交叉韧带切除法制作KOA模型,手术6周后四妙散组以四妙散药液灌胃,正常对照组和KOA模型组以生理盐水灌胃。连续灌胃4周后取膝关节软骨组织,采用Annexin V-FITC/PI双染流式细胞术测定凋亡细胞所占比例,采用荧光定量PCR检测关节软骨组织中凋亡和自噬相关基因表达水平。结果:与正常对照组比较,KOA模型组大鼠关节软骨细胞凋亡率显著升高,关节软骨细胞中凋亡相关蛋白Fadd、Casp3的表达显著升高,自噬相关蛋白LC3的表达显著降低,差异均有统计学意义(P<0.05)。与KOA模型组比较,四妙散组大鼠关节软骨细胞凋亡率显著降低,关节软骨细胞中凋亡相关蛋白Fadd、Casp3的表达显著降低,自噬相关蛋白LC3的表达显著升高,差异均有统计学意义(P<0.05)。结论:软骨细胞凋亡是KOA发病中的重要因素,该凋亡可能通过FADD-CASP3途径发挥作用。软骨细胞自噬作用的激活可抑制细胞凋亡,可能是延缓及控制KOA进展的一个新机制,四妙散可能通过该机制发挥治疗作用。
Objective: To study regulation effect of Simiao powder on apoptosis and autophagy in knee osteoarthritis( KOA)rat models. Methods: Selected 27 SD rats of 8 week old, and divided them into normal control group, KOA model group and Simiao powder group randomly. Anterior cruciate ligament(ACL) resectional therapy was applied to the KOA model group and Simiao powder group to establish KOA model. Six weeks after operation, Simiao powder group was given herb liquor of Simiao powder by gavage, while the normal control group and KOA model group were given saline by gavage. Knee joint cartilage tissue was taken after continuous gavage for four weeks. Detected proportion of apoptotic cell with Annexin V-FITC/PI double staining flow cytometry, and detected expression level of apoptosis and autophagy related gene in joint cartilage tissue with fluorescence quantitative PCR. Results: Compared with normal control group, apoptosis rate of chondrocytes of KOA model group was increased significantly, and expression of FAS-associated death domain(Fadd) and caspase-3(Casp3) in joint cartilage tissue was increased significantly, while expression of autophagy associated protein 1 light chain 3 was decreased significantly, differences being significant(P<0.05). Compared with KOA model group, apoptosis rate of chondrocytes in Simiao powder group was decreased significantly, and expression of Fadd and Casp3 in joint cartilage tissue was decreased significantly, while expression of autophagy associated protein 1 light chain 3 was increased significantly, differences being significant(P <0.05). Conclusion: Apoptosis of chondrocytes is one of the major causes of KOA, which may work by FADD-CASP3 pathway. Activation of autophagocytosis of chondrocytes may inhibit apoptosis, and it may be a new mechanism that Simiao powder functions for delaying and controlling the progression of KOA.
Objective: To study regulation effect of Simiao powder on apoptosis and autophagy in knee osteoarthritis( KOA)rat models. Methods: Selected 27 SD rats of 8 week old, and divided them into normal control group, KOA model group and Simiao powder group randomly. Anterior cruciate ligament(ACL) resectional therapy was applied to the KOA model group and Simiao powder group to establish KOA model. Six weeks after operation, Simiao powder group was given herb liquor of Simiao powder by gavage, while the normal control group and KOA model group were given saline by gavage. Knee joint cartilage tissue was taken after continuous gavage for four weeks. Detected proportion of apoptotic cell with Annexin V-FITC/PI double staining flow cytometry, and detected expression level of apoptosis and autophagy related gene in joint cartilage tissue with fluorescence quantitative PCR. Results: Compared with normal control group, apoptosis rate of chondrocytes of KOA model group was increased significantly, and expression of FAS-associated death domain(Fadd) and caspase-3(Casp3) in joint cartilage tissue was increased significantly, while expression of autophagy associated protein 1 light chain 3 was decreased significantly, differences being significant(P<0.05). Compared with KOA model group, apoptosis rate of chondrocytes in Simiao powder group was decreased significantly, and expression of Fadd and Casp3 in joint cartilage tissue was decreased significantly, while expression of autophagy associated protein 1 light chain 3 was increased significantly, differences being significant(P <0.05). Conclusion: Apoptosis of chondrocytes is one of the major causes of KOA, which may work by FADD-CASP3 pathway. Activation of autophagocytosis of chondrocytes may inhibit apoptosis, and it may be a new mechanism that Simiao powder functions for delaying and controlling the progression of KOA.
引文
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[2]Battistelli M,Salucci S,Olivotto E,et al.Cell death in human articular chondrocyte:a morpho-functional study in micromass model[J].Apoptosis,2014,19(10):1471-1483.
[3]Beyer C,Zampetaki A,Lin NY,et al.Signature of circulating microRNAs in osteoarthritis[J].Ann Rheum Dis,2015,74(3):e18.
[4]Maiuri MC,Zalckvar E,Kimchi A,et al.Self-eating and self-killing:crosstalk between autophagy and apoptosis[J].Nat Rev Mol Cell Biol,2007,8(9):741-752.
[5]Kroemer G,Marino G,Levine B.Autophagy and the integrated stress response[J].Mol Cell,2010,40(2):280-293.
[6]Carames B,Taniguchi N,Otsuki S,et al.Autophagy is a protective mechanism in normal cartilage,and its aging-related loss is linked with cell death and osteoarthritis[J].Arthritis Rheum,2010,62(3):791-801.
[7]Carames B,Hasegawa A,Taniguchi N,et al.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis[J].Ann Rheum Dis,2012,71(4):575-581.
[8]王婧,张忠辉,孙娇梦,等.大鼠骨关节炎三种动物模型的建立及比较[J].中国细胞生物学学报,2010,32(3):456-460.
[9]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[10]Takayama K,Kawakami Y,Lee S,et al.Involvement of ERCC1 in the pathogenesis of osteoarthritis through the modulation of apoptosis and cellular senescence[J].J Orthop Res,2014,32(10):1326-1332.
[11]Wang F,Wu L,Li L,et al.Monotropein exerts protective effects against IL-1β-induced apoptosis and catabolic responses on osteoarthritis chondrocytes[J].Int Immunopharmacol,2014,23(2):575-580.
[12]Lopez-Armada MJ,Carames B,Lires-Dean M,et al.Cytokines,tumor necrosis factor-alpha and interleukin-1beta,differentially regulate apoptosis in osteoarthritis cultured human chondrocytes[J].Osteoarthritis Cartilage,2006,14(7):660-669.
[13]李慧英,孟东方,苏建光.四妙散干预兔膝关节创伤性滑膜炎白细胞介素-1β和肿瘤坏死因子-α水平的研究[J].中华中医药杂志,2013,28(10):3043-3045.
[14]赵鹏飞,汪利合,李慧英,等.加味四妙散对兔膝骨关节炎il-6和bfgf的影响[J].中医学报,2015,30(8):1166-1169.
[15]阮丽萍,刘健,葛瑶,等.骨关节炎大鼠软骨PI3K/AktmTOR及Beclin-1自噬通路的表达及相关性分析[J].华中科技大学学报:医学版,2015,44(4):429-433,439.