中国骨质疏松杂志

补肾活血方抑制骨关节炎大鼠软骨细胞凋亡的机制

The mechanism of Bushen Huoxue formula in the inhibition of chondrocyte apoptosis in rats with osteoarthritis

DOI：10.3969/j.issn.1006-7108.2025.03.010

英文关键词:Bushen Huoxue formula osteoarthritis chondrocytes apoptosis

基金项目:湖南省中医药科研计划项目（D2022114）；湖南省自然科学基金省市联合项目（2022JJ50049）

作者	单位
胡宏志李娟毛滔*	湖南中医药大学第二附属医院骨伤四科，湖南长沙 410005

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中文摘要:

目的探讨补肾活血方（BSHX）抑制骨关节炎大鼠软骨细胞凋亡的作用机制。方法将CHON-001细胞随机分为空白组、LPS组（100 ng/mL）、BSHX组（100 ng/mL LPS+20 % BSHX含药血清）、BSHX+DMSO组（100 ng/mL LPS+20 % BSHX含药血清+0.1 % DMSO）、BSHX+Derivative 83组（100 ng/mL LPS+20 % BSHX含药血清+120 μmol/L Derivative 83）。分析各组细胞增殖、凋亡以及上清液中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6、IL-1β水平；大鼠随机分为假手术组、模型组、BSHX组，检测血清中一氧化氮合成酶（INOS）、IL-6、环氧合酶2（COX2）水平以及软骨组织中Collage Ⅱ、Aggrecan、ADAMTS-5 mRNA表达水平；并对软骨组织及细胞中Cleaved caspase-3、Bax、Bcl-2及Wnt3a/β-catenin蛋白表达水平进行分析。结果与空白组相比，LPS组细胞凋亡率、TNF-α、IL-6、IL-1β水平、Cleaved caspase-3、Bax、Wnt3a、β-catenin、p-NF-kB p65/NF-kB p65蛋白表达显著增加，24 h、48 h OD450值以及EdU阳性率、Bcl-2表达明显降低（P<0.05）；BSHX改善了LPS诱导的CHON-001细胞上述指标（P<0.05），但Derivative 83逆转了BSHX对LPS诱导的CHON-001细胞的保护作用（P<0.05）。动物实验结果显示，与假手术组相比，模型组INOS、IL-6、COX2水平、ADAMTS-5 mRNA、Cleaved caspase-3、Bax、Wnt3a、β-catenin、p-NF-kB p65/NF-kB p65蛋白表达显著增加，Collage Ⅱ mRNA、Aggrecan mRNA、Bcl-2表达明显降低（P<0.05）；与模型组相比，BSHX组INOS、IL-6、COX2水平、ADAMTS-5 mRNA、Cleaved caspase-3、Bax、Wnt3a、β-catenin、p-NF-kB p65/NF-kB p65蛋白表达显著降低，Collage Ⅱ mRNA、Aggrecan mRNA、Bcl-2表达明显增加（P<0.05）。结论 BSHX抑制骨关节炎大鼠软骨细胞凋亡。

英文摘要:

Objective To investigate the mechanism of Bushen Huoxue formula (BSHX) in inhibiting apoptosis of chondrocytes in rats with osteoarthritis. Methods CHON-001 cells were randomly grouped into blank group, LPS group (100 ng/mL), BSHX group (100 ng/mL LPS + 20% BSHX medicated serum), BSHX+DMSO group (100 ng/mL LPS + 20% BSHX medicated serum + 0.1% DMSO), and BSHX+Derivative 83 group (100 ng/mL LPS + 20% BSHX medicated serum + 120 μmol/L Derivative 83). The proliferation and apoptosis of cells, and the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in the supernatant in each group were analyzed. The rats were randomly divided into sham operation group, model group, and BSHX group. The serum levels of nitric oxide synthase (INOS), IL-6, and cyclooxygenase 2 (COX2), and the mRNA expression levels of Collage II, Aggrecan and ADAMTS-5 in cartilage tissue were measured. The expression levels of Cleaved caspase-3, Bax, Bcl-2, and Wnt3a/β-catenin in the cartilage and cells were analyzed. Results Compared to those in the blank group, the apoptosis rate, TNF-α, IL-6, IL-1β levels, Cleaved caspase-3, Bax, Wnt3a, β-catenin, and p-NF-kB p65/NF-kB p65 protein expressions in the LPS group increased obviously. The OD450 value at 24h and 48h, the positive rate of EdU, and the expression of Bcl-2 decreased obviously (P<0.05). BSHX improved the above indexes in LPS-induced CHON-001 cells (P<0.05). Derivative 83 reversed the protective effect of BSHX on LPS-induced CHON-001 cells (P<0.05). The results of animal experiments showed that compared to those in the sham group, the protein expressions of INOS, IL-6, and COX2, ADAMTS-5 mRNA, Cleaved caspase-3, Bax, Wnt3a, β-catenin, and p-NF-kB p65/NF-kB p65 in the model group increased obviously, and the expressions of collage II mRNA, aggrecan mRNA, and Bcl-2 decreased obviously (P<0.05). Compared to those in the model group, the protein expressions of INOS, IL-6, COX2, ADAMTS-5 mRNA, Cleaved caspase-3, Bax, Wnt3a, β-catenin, and p-NF-kB p65/NF-kB p65 decreased obviously in the BSHX group, the expressions of collage II mRNA, aggrecan mRNA and Bcl-2 increased obviously (P<0.05). Conclusion BSHX inhibits chondrocyte apoptosis in osteoarthritis rats.

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