Mechanism of Duhuo Jisheng Decoction for treating knee osteoarthritis

doi:10.3969/j.issn.2095-4344.1241

Abstract

Abstract:

BACKGROUND: Preliminary study has shown that Duhuo Jisheng Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear.
OBJECTIVE: To observe the effect of Duhuo Jisheng Decoction on the related regulatory factors in miR-146a/-155-NF-κB/p38 MAPK signaling pathway in cartilage and synovium of rats with knee osteoarthritis.
METHODS: Thirty-six male rats were provided by Shanghai Slack Laboratory Animals Co., Ltd. The study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine, approval number: [2018] (014). After 1 week of acclimation, the rats were randomly divided into blank group (n=12) and model group (n=24). Osteoarthritis model was induced in rat right knees by modified Hulth method. One week later, the rat models were randomly divided into model control group (n=12) and experiment group (n=12). The blank and model control groups received an equivalent amount of normal saline (10 mL/kg•d) intragastrically. The experiment group received a clinical oral dose of Duhuo Jisheng Decoction (9.3 g/kg•d) intragastrically. After three months of treatment, the animals were sacrificed and synovial tissue and cartilage tissue were obtained. The contents of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13 were measured by ELISA. The protein levels of nuclear factor-κB p65 and p38 mitogen-activated protein kinase were detected by immunohistochemistry. The mRNA expression levels of miR-146a, miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase were tested by real-time PCR.
RESULTS AND CONCLUSION: (1) Real-time PCR results suggest that Duhuo Jisheng Decoction could inhibit the mRNA expression of miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase, and promote miR-146a mRNA expression (P < 0.01). (2) The results of immunohistochemistry were in agreement with the results of real-time PCR, indicating that Duhuo Jisheng Decoction could inhibit the protein expression of nuclear factor-κB p65 and p38 mitogen-activated protein kinase (P < 0.01). (3) ELISA results manifest that Duhuo Jisheng Decoction could effectively inhibit the expression of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13. (4) These results suggest that Duhuo Jisheng Decoction can inhibit the inflammatory reaction in knee osteoarthritis by regulating the miR-146a/-155-NF-κB/p38 MAPK signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee osteoarthritis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Duhuo Jisheng Decoction, knee osteoarthritis, nuclear factor-κB signaling pathway, p38 MAPK signaling pathway, nitric oxide synthase, cyclooxygenase 2, matrix metalloproteinase 3, matrix metalloproteinase 13

CLC Number:

R496
R318

Wu Guangwen1, Liu Shuru2, Chen Jun3, Lin Jie1, Zhao Zhongsheng1, Ye Jinxia1. Mechanism of Duhuo Jisheng Decoction for treating knee osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(19): 2965-2971.

Figures/Tables 2

2.1 实验动物数量分析实验选用大鼠36只，分为3组，实验过程中，模型组麻醉意外致死1只，实验组在灌胃过程中出现腹泻致死1只，均给予补全后，36只大鼠进入结果分析。 2.2 大鼠关节软骨组织miR-146a、miR-155的表达与空白组相比，模型组miR-146a表达下降到0.092 8±0.012 1，miR-155表达升高到1.950 7±0.052 7；实验组miR-146a表达下降到0.763 0±0.032 7，miR-155表达升高到 1.468 8±0.062 8，差异均有非常显著性意义(P < 0.01)。与模型组相比，实验组miR-146a表达显著上升，miR-155表达显著下降均P < 0.01)。见图1。 2.3 大鼠关节软骨组织NF-κB p65和p38 MAPK mRNA的表达与空白组相比，模型组NF-κB p65表达升高到 1.515 2±0.068，p38 MAPK表达升高到1.854 3±0.068 9，差异均有显著性意义(P < 0.01)；实验组NF-κB p65表达升高到1.147 2±0.053 3，p38 MAPK表达升高到1.311 8± 0.034 8，差异均有显著性意义(P < 0.05或P < 0.01)。与模型组相比，实验组NF-κB p65、p38 MAPK表达均显著下降(P < 0.01)。见图2。 2.4 大鼠关节软骨组织NF-κB p65和p38 MAPK蛋白的表达 NF-κB p65、p38 MAPK阳性染色呈棕黄色或棕褐色，见图3，4。比较各组阳性率，可见各组对NF-κB p65、p38 MAPK蛋白表达的影响与其对NF-κB p65、p38 MAPK mRNA的影响具有相同的趋势，与空白组相比，模型组NF-κB p65、p38 MAPK阳性率显著升高(P < 0.01)；与模型组相比，实验组NF-κB p65、p38 MAPK阳性率均显著降低(P < 0.01)。见图3D，图4D。 2.5 大鼠滑膜组织诱导型一氧化氮合酶、环氧合酶2、基质金属蛋白酶3和基质金属蛋白酶13水平与空白组相比，模型组和实验组诱导型一氧化氮合酶、环氧合酶2、基质金属蛋白酶3和基质金属蛋白酶13水平均显著升高(P < 0.01)；与模型组相比，实验组上述4项指标检测值均显著降低(P < 0.01)。见图5。"

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