桃仁-红花通过抑制NF-κB信号通路改善IL-1β诱导的软骨炎症和降解

Persicae Semen-Carthami Flos Ameliorates IL-1β-Induced Cartilage Inflammation and Degradation by Inhibiting NF-κB Signaling Pathway

目的应用网络药理学分析桃仁-红花治疗膝骨关节炎(KOA)的潜在作用机制,并通过体内和体外实验验证。方法通过网络药理学筛选桃仁-红花治疗膝骨关节炎的可能活性成分及作用机制。体外实验:提取小鼠原代软骨细胞,CCK-8法筛选桃仁-红花冻干粉干预软骨细胞的最佳浓度。随后,软骨细胞随机分为对照组、IL-1β组、桃仁-红花低剂量组、桃仁-红花中剂量组和桃仁-红花高剂量组。10 ng·mL^(-1)的IL-1β用于模拟KOA炎症环境。qRT-PCR用于检测软骨细胞环氧化物酶2(COX-2)、一氧化氮合酶(iNOS)和IL-6的mRNA表达。Western Blot用于检测软骨细胞Ⅱ型胶原蛋白(Collagen Ⅱ)、聚集蛋白聚糖(Aggrecan)和高迁移率族框9(Sox9)的蛋白水平和NF-κB信号通路的相关标志物。此外,免疫荧光用于检测p65在细胞内的定位和表达。体内实验:采用内侧半月板失稳术(DMM)构建膝骨关节炎小鼠模型。将30只小鼠随机分为3组:假手术组、模型组和给药组。HE、番红O/快速绿染色和OARSI评分用于评估小鼠的关节损伤。结果筛选出30个桃仁-红花治疗膝骨关节炎的有效活性成分和126个靶点。潜在成分主要包括槲皮素、β-谷甾醇、鞣花酸、天竺葵素和豆甾醇等。分子对接结果表明,活性成分与核心靶点具有良好的结合活性。实验研究表明,桃仁-红花可降低IL-1β诱导的COX-2、iNOS和IL-6 mRNA的上调,并增加IL-1β诱导的Collagen Ⅱ、Aggrecan和Sox9的蛋白表达下降,降低小鼠软骨细胞中IL-1β诱导的p65磷酸化和p65核转位,以200μg·mL^(-1)组效果为佳(P<0.05)。此外,桃仁-红花改善了膝骨关节炎小鼠的关节退变(P<0.05)。结论研究表明,桃仁-红花通过抑制NF-κB信号通路改善软骨细胞基质降解和炎症状态。桃仁-红花在膝骨关节炎的发展中起着积极的治疗作用,为其进一步的基础研究和临床应用提供了新的思路。

Objective To analyze the potential mechanism of Persicae Semen and Carthami Flos in the treatment of knee osteoarthritis(KOA)by network pharmacology,and to verify the results by in vivo and in vitro experiments.Methods The possible active components and mechanism of action of Persicae Semen and Carthami Flos in the treatment of KOA were screened by network pharmacology.In vitro experiment was designed as follow:the mouse primary chondrocytes were extracted,and the CCK-8 was used to screen the optimal concentration of Persicae Semen and Carthami Flos freeze-dried powder to interfere with chondrocytes.Subsequently,chondrocytes were randomly divided into control group,IL-1β group,Persicae Semen and Carthami Flos low-,medium-and highdose groups.10 ng·mL^(-1)of IL-1β was used to simulate the inflammatory environment of KOA.qRT-PCR was used to detect the mRNA expressions of cyclooxygenase 2(COX-2),nitric oxide synthase(iNOS)and IL-6 in chondrocytes.Western Blot was used to detect the protein levels of type Ⅱ collagen(Collagen Ⅱ),Aggrecan and SRY-type high mobility group box 9(Sox9)in chondrocytes and related markers of NF-κB signaling pathway.In addition,immunofluorescence was used to detect the localization and expression of p65 in cells.In vivo experiment was designed as follow:a mouse model of KOA was established by destabilization of medial meniscus(DMM).Thirty mice were randomly divided into three groups:sham-operation group,DMM group and DMM+Persicae Semen and Carthami Flos group.HE,Safranin O/fast green staining and OARSI scores were used to assess joint damage in mice.Results Thirty active components and 126 targets were screened out in Persicae Semen and Carthami Flos treatment of KOA.The potential components mainly included quercetin,beta sitosterol,ellagic acid,pelargonidin and stigmasterol.Molecular docking results showed that the active components had good binding activity to the core targets.Experimental studies showed that Persicae Semen and Carthami Flos decreased IL-1β-induced mRNA up-regulation of COX-2,iNOS and IL-6,increased IL-1β-induced down-regulation on protein expression of Collagen Ⅱ,Aggrecan and Sox9,and reduced IL-1β-induced p65 phosphorylation and p65 nuclear translocation in mouse chondrocytes,especially the 200μg·mL^(-1)group(P<0.05).Furthermore,Persicae Semen and Carthami Flosimproved joint degeneration in KOA mice(P<0.05).Conclusion Our study showed that Persicae Semen and Carthami Flos improves cartilage matrix degradation and inflammatory state by inhibiting NF-κB signaling pathway.Persicae Semen and Carthami Flos plays an active therapeutic role in the development of KOA,which provides new ideas for further basic research and clinical applications.