基于HMGB1/TLR4/NF-κB信号通路研究藁本内酯衍生物抑制IL-1β诱导的大鼠软骨细胞凋亡和炎症反应的作用机制

Ligusticum cycloprolactam inhibits IL-1β-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway

软骨细胞是关节软骨中的独特驻留细胞类型,当其发生一系列病理改变时能够导致骨关节炎(osteoarthritis,OA)的发生。藁本内酯衍生物(ligusticum cycloprolactam,LIGc)是中药当归药效标志物藁本内酯(Z-ligustilide,LIG)的衍生物,具有抗炎、抑制细胞凋亡等多种生物学功能。然而,其对OA软骨细胞的保护作用以及潜在的作用机制尚不清楚。该研究拟进行体外实验探索LIGc抗OA保护软骨细胞作用的分子机制。采用白细胞介素-1β(IL-1β)诱导建立大鼠OA软骨细胞炎症模型,并单独使用LIGc和联合高迁移率族蛋白B1/Toll样受体4/核因子-κB(HMGB1/TLR4/NF-κB)信号通路阻断剂甘草酸(glycyrrhizic acid,GA)进行干预,以系统评估其治疗效果。通过cell counting kit-8(CCK-8)试剂盒检测不同浓度下LIGc对软骨细胞活力的影响,并筛选出最佳作用浓度;Annexin V-FITC/PI凋亡染色检测各组软骨细胞凋亡情况;酶联免疫吸附法(ELISA)测定各组软骨细胞上清中环氧合酶-2(COX-2)、前列腺素-2(PGE2)、肿瘤坏死因子-α(TNF-α)的表达;蛋白质印迹法(Western blot)检测各组软骨细胞B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、HMGB1、TLR4、NF-κB p65的蛋白表达水平;实时荧光定量PCR(RT-qPCR)检测各组软骨细胞HMGB1、TLR4、NF-κB p65、髓样分化因子88(MyD88)的基因表达变化。通过CCK-8确定了LIGc对软骨细胞的大致安全浓度范围,并进一步明确了LIGc作用的最佳浓度。在IL-1β的干预下,成功构建大鼠OA软骨细胞模型,且在该细胞模型中,软骨细胞凋亡率明显增加。与此同时,ELISA检测该组软骨细胞上清中COX-2、PGE2、TNF-α的表达均显著上升;Western blot检测发现模型组软骨细胞Bax、caspase-3、HMGB1、TLR4、NF-κB p65的蛋白表达显著上调,Bcl-2的蛋白表达受到明显抑制;RT-qPCR测定显示软骨细胞内HMGB1、TLR4、NF-κB p65、MyD88的基因表达经IL-1β干预后均有所提高。然而,随着LIGc的加入逆转了IL-1β诱导下上述一系列因子的表达变化。此外,LIGc联合GA共同作用下较单独添加LIGc对实验结果的逆转趋势更为明显。这些研究结果表明,从中药当归中提取并衍生的LIGc能够起到抑制软骨细胞炎症反应及减少软骨细胞凋亡的作用,且该作用的发挥可能与HMGB1/TLR4/NF-κB信号通路具有较强的相关性。LIGc保护软骨细胞的药理作用,对于延缓OA病情及改善患者临床症状具有潜在价值,值得进一步深入研究。

Chondrocytes are unique resident cells in the articular cartilage,and the pathological changes of them can lead to the occurrence of osteoarthritis(OA).Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG),a pharmacodynamic marker of Angelica sinensis,which has various biological functions such as anti-inflammation and inhibition of cell apoptosis.However,its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear.This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA.The inflammation model of rat OA chondrocyte model was established by using interleukin-1β(IL-1β) to induce.LIGc alone and combined with glycyrrhizic acid(GA),a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway,were used to intervene in the model,and the therapeutic effects were systematically evaluated.The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8),and the optimal LIGc concentration was screened out.Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group.The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2),prostaglandin-2(PGE2),and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group.Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3,HMGB1,TLR4,and NF-κB p65.The mRNA levels of HMGB1,TLR4,NF-κB p65,and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR).The safe concentration range of LIGc on chondrocytes was determined by CCK-8,and then the optimal concentration of LIGc for exerting the effect was clarified.Under the intervention of IL-1β,the rat chondrocyte model of OA was successfully established.The modeled chondrocytes showed increased apoptosis rate,promoted expression of COX-2,PGE2,and TNF-α,up-regulated protein levels of Bax,caspase-3,HMGB1,TLR4,and NF-κB p65 and mRNA levels of HMGB1,TLR4,NF-κB p65,and MyD88,and down-regulated protein level of Bcl-2.However,LIGc reversed the IL-1β-induced changes of the above factors.Moreover,LIGc combined with GA showed more significant reversal effect than LIGc alone.These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A.sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes,and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway.The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients,and deserves further study.