槲皮素通过激活自噬对LPS诱导的软骨细胞基质代谢及炎症的影响

Effect of Quercetin on LPS-induced Chondrocyte Matrix Metabolism and Inflammation by Activating Autophagy

目的:从细胞自噬角度探讨槲皮素调控膝骨关节炎(KOA)软骨细胞外基质代谢及炎症反应的作用机制。方法:提取软骨细胞、传代培养,及用Ⅱ型胶原蛋白(CollagenⅡ)免疫荧光染色鉴定原代细胞;将脂多糖(LPS)诱导的软骨细胞分为空白组(不做任何处理)、模型组(10 mg·L^(-1)LPS处理48 h)、槲皮素低、中、高剂量组(10 mg·L^(-1)LPS处理48 h+50、100、150 mmol·L^(-1)槲皮素处理24 h)。细胞增殖与活性检测(CCK-8)法检测LPS(2.5、5、7.5、10、12.5 mg·L^(-1))对软骨细胞不同时间(24、48、72 h)增殖的抑制作用;槲皮素(50、100、150、200 mmol·L^(-1))对LPS诱导的软骨细胞不同时间(12、24、48 h)增殖的影响;蛋白免疫印迹法(Western blot,WB)检测微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)和泛素结合蛋白p62(p62)蛋白表达。用3-甲基腺嘌呤(3-MA)干预LPS诱导的软骨细胞,分为空白组(不做任何处理)、模型组(10 mg·L^(-1)LPS)、槲皮素组(模型组+100 mmol·L^(-1)槲皮素)、3-MA组(模型组+100μmol·L^(-1)3-MA)、3-MA+槲皮素组(模型组+100μmol·L^(-1)3-MA+100 mmol·L^(-1)槲皮素),先用LPS处理48 h后,3-MA处理2 h,再用槲皮素干预24 h。酶联免疫吸附测定法(ELISA)检测白细胞介素-1β(IL^(-1)β)、肿瘤坏死因子-α(TNF-α)含量;WB检测基质金属蛋白酶-13(MMP-13)、金属蛋白酶组织抑制因子1(TIMP1)蛋白表达。结果:CollagenⅡ免疫荧光鉴定结果示,所提取的细胞符合软骨细胞特征;CCK-8法筛选LPS最佳造模质量浓度为10 mg·L^(-1)、48 h,槲皮素最佳浓度为100 mmol·L^(-1)、24 h;WB结果示,与空白组比较,模型组LC3Ⅱ表达显著降低(P<0.01),p62表达显著升高(P<0.01),与模型组比较,槲皮素低、中、高剂量组LC3Ⅱ表达显著升高(P<0.01),其槲皮素中剂量组最显著,p62表达显著降低(P<0.01),其槲皮素中剂量组最显著;与空白组比较,模型组MMP-13表达明显升高(P<0.05),TIMP1表达显著降低(P<0.01);与模型组比较,槲皮素组、3-MA+槲皮素组MMP-13表达明显降低(P<0.05,P<0.01),其中槲皮素组最显著,TIMP1表达显著升高(P<0.01),其中槲皮素组最显著。倒置显微镜下观察软骨细胞形态学改变结果示,槲皮素可恢复受损的软骨细胞形态;CCK-8检测各组细胞增殖结果示,与空白组比较,模型组软骨细胞增殖明显被抑制(P<0.01);与模型组比较,槲皮素组、3-MA+槲皮素组软骨细胞增殖显著升高(P<0.01),其中槲皮素组最显著;ELISA检测结果示,与空白组比较,模型组IL^(-1)β、TNF-α含量显著升高(P<0.01);与模型组比较,槲皮素组、3-MA+槲皮素组IL^(-1)β、TNF-α含量明显降低(P<0.05,P<0.01),其中槲皮素组降低最显著。结论:槲皮素可促进LPS诱导的软骨细胞增殖,调控软骨细胞外基质合成与代谢平衡,抑制炎症反应,恢复软骨细胞功能,其机制可能与槲皮素激活细胞自噬有关。

Objective: To investigate the mechanism of quercetin in regulating chondrocyte extracellular matrix metabolism and inflammatory response in knee osteoarthritis(KOA)from the perspective of autophagy. Method: Chondrocytes were extracted and cultured,and the primary cells were identified by immunofluorescence staining with collagen Ⅱ. The chondrocytes induced by lipopolysaccharide(LPS)were divided into a control group(without any treatment),a model group(10 mg·L-1LPS treatment for 48 h),and low-,medium-,and high-dose quercetin group(10 mg·L^(-1) LPS treatment for 48 h combined with 50,100,and150 mmol·L^(-1) quercetin for 24 h). The inhibitory effects of LPS(2.5,5,7.5,10,12.5 mg·L-1)on the proliferation of chondrocytes for different periods(24,48,72 h)were detected by cell counting kit-8(CCK-8).The effects of quercetin(50,100,150,200 mmol·L^(-1)) on the LPS-induced proliferation of chondrocytes for different periods(12,24,and 48 h)were investigated. The expression of microtubule-associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ)and ubiquitin-binding protein p62 was detected by Western blot. LPS-induced chondrocytes were treated with 3-methyladenine(3-MA). The resultant cells were divided into a control group(without any treatment),a model group(10 mg·L-1LPS),a quercetin group(model group + 100 mmol·L^(-1) quercetin),a3-MA group(model group + 100 μmol·L-13-MA), and a 3-MA + quercetin group(model group +100 μmol·L-13-MA + 100 mmol·L-1quercetin,specifically,LPS for 48 h,3-MA for 2 h,and then quercetin for24 h). The content of interleukin(IL)-1β and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of matrix metalloproteinase 13(MMP-13)and tissue inhibitor of metalloproteinase 1(TIMP1) was detected by Western blot. Result: Collagen Ⅱimmunofluorescence staining showed that the extracted cells were consistent with the characteristics of chondrocytes. As revealed by CCK-8,the optimum concentration of LPS was 10 mg·L-1with an action time of48 h,and the optimum concentration of quercetin was 100 mmol·L-1with an action time of 24 h. Western blot results showed that compared with the control group,the model group showed decreased expression of LC3 Ⅱ(P<0.01)and increased expression of p62(P<0.01). The expression of LC3 Ⅱ in the quercetin groups was higher than that in the control group(P<0.01),especially in the medium-dose quercetin group. The p62expression in the quercetin groups was lower than that in the control group(P<0.01),especially in the mediumdose quercetin group. Compared with the control group,the model group showed increased expression of MMP-13(P<0.05) and decreased expression of TIMP1(P<0.01). Compared with the model group, the quercetin groups and the 3-MA + quercetin group showed decreased expression of MMP-13(P<0.05,P<0.01),especially the quercetin groups,and increased expression of TIMP1(P<0.01),especially the quercetin groups.Morphological changes in chondrocytes under the inverted microscope showed that quercetin could restore the morphology of damaged chondrocytes. CCK-8 showed that compared with the control group,the model group showed inhibited chondrocyte proliferation(P<0.01),and compared with the model group,the quercetin groups and the 3-MA + quercetin group showed promoted chondrocyte proliferation(P<0.01),especially the quercetin groups. ELISA results showed that IL-1β and TNF-α levels in the model group were higher than those in the control group(P<0.01),and the levels of IL-1β and TNF-α in the quercetin groups and the 3-MA + quercetin group were lower than those in the model group(P<0.05,P<0.01),and the decrease in the quercetin groups was the most significant. Conclusion: Quercetin can promote LPS-induced chondrocyte proliferation,regulate chondrocyte extracellular matrix synthesis and metabolic balance,inhibit the inflammatory response,and restore chondrocyte function. The mechanism may be related to the activation of autophagy by quercetin.