黄芩素通过Sirt1抑制成纤维样滑膜细胞衰老缓解骨关节炎的作用机制

Mechanism of baicalein in alleviating osteoarthritis by inhibiting fibroblast-like synoviocytes senescence through Sirt1

目的:探讨黄芩素通过调控Sirtuin(Sirt)抑制成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)衰老,缓解骨关节炎的机制。方法:提取非骨关节炎患者(3例)及骨关节炎患者(3例)滑膜中的FLS,通过Western blot、β-半乳糖苷酶染色和免疫荧光等方法评估骨关节炎患者FLS的衰老程度,并检测FLS中Sirt家族蛋白和mRNA的变化水平。通过转染Sirt1 siRNA敲低FLS中Sirt1的相对表达水平,分析FLS抗氧化能力及衰老程度。将软骨细胞与FLS共培养,通过Western blot检测软骨细胞功能变化。使用博莱霉素(Bleomycin,BLM)诱导FLS衰老后予以不同浓度黄芩素处理,检测Sirt1、p16、p21蛋白表达水平,并检测细胞内活性氧(reactive oxygen species,ROS)表达水平。敲低FLS中Sirt1的表达水平并予以BLM和黄芩素处理,采用Western blot和ROS检测Sirt1和ROS相对表达量。BLM诱导FLS衰老后加入黄芩素和Compound C,通过Western blot、ROS检测磷酸化腺苷酸活化蛋白激酶(phosphorylated AMP protein-activated kinase,pAMPK)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)、p16、p21和ROS相对表达量。结果:骨关节组FLS的p16和p21相对表达量为3.66±0.38和3.55±0.34,高于非骨关节炎组的1.00±0.07、1.00±0.09(t=11.860,P<0.001;t=12.520,P<0.001)。骨关节炎组FLS中Sirt1和Sirt6的相对表达水平为0.30±0.04和0.16±0.01,小于正常组的1.00±0.03和1.00±0.04(t=23.840,P<0.001;t=34.130,P<0.001)。黄芩素处理后,BLM组、BLM+Bai组和对照组的ROS相对表达量分别为2.58±0.28、1.65±0.14和1.00±0.24,组间差异有统计学意义(F=35.700,P<0.001),其中BLM组和BLM+Bai组均大于对照组,BLM+Bai组低于BLM组(P<0.05)。Compound C+黄芩素处理后,pAMPK及NRF2相对表达为0.28±0.02和0.38±0.09,低于BLM+Bai组的0.56±0.07和0.60±0.08(P<0.05)。ROS相对表达量由1.75±0.16升高至3.45±0.12(P<0.001)。BLM+Bai+Compound C组、BLM+Bai组和对照组β-半乳糖苷酶阳性比例分别为47.30%±4.29%、18.18%±3.89%和7.70%±3.53%(F=109.700,P<0.001),其中BLM+Bai+Compound C组高于BLM+Bai组(P<0.05)。结论:骨关节炎患者Sirt1表达下调导致FLS衰老,加速骨关节炎进展。黄芩素可通过激活Sirt1/AMPK/NRF2通路抑制FLS衰老,可能延缓骨关节炎进展,进而改善软骨细胞功能。

Objective To investigate the mechanism by which baicalein alleviates osteoarthritis by regulating Sirtuin(Sirt)to inhibit the senescence of fibroblast-like synoviocytes(FLS).Methods FLS were extracted from synovium of non-osteoarthritis patients(3 patients)and osteoarthritis patients(3 patients),and assessed the senescence of FLS in osteoarthritis patients by Western blot,β-galactosidase staining and immunofluorescence,and examined the changing levels of Sirt family proteins and mRNAs in FLS.The relative expression level of Sirt1 in FLS was knocked down by transfection with Sirt1 siRNA,and the antioxidative capacity and senescence degree of FLS were analyzed.Chondrocytes were co-cultured with FLS and functional changes in chondrocytes were detected by Western blot.After inducing FLS senescence with bleomycin(BLM),different concentrations of baicalein were administered to detect the expression levels of Sirt1,p16,and p21 proteins and intracellular reactive oxygen species(ROS).The expression level of Sirt1 in FLS was knocked down,and BLM and baicalein treatments were applied.The relative expression levels of Sirt1 and ROS were detected using Western blot and ROS assays.After BLM-induced FLS senescence,baicalein and Compound C were added.The relative expression levels of phosphorylated AMP-activated protein kinase(pAMPK),nuclear factor erythroid 2-related factor 2(NRF2),p16,p21,and ROS were detected by Western blot and ROS assays.Results The relative expression of p16 and p21 in FLS in the osteoarthritic group were 3.66±0.38 and 3.55±0.34,which were higher than those in non-osteoarthritis group 1.00±0.07 and 1.00±0.09(t=11.860,P<0.001;t=12.520,P<0.001).The relative expression levels of Sirt1 and Sirt6 in FLS in the osteoarthritis group were 0.30±0.04 and 0.16±0.01,which were smaller than those in the non-osteoarthritis group 1.00±0.03 and 1.00±0.04(t=23.840,P<0.001;t=34.130,P<0.001).After baicalein treatment,the relative expression of ROS was 2.58±0.28,1.65±0.14 and 1.00±0.24 in the BLM,BLM+Bai and control groups,respectively,with statistically significant differences between the groups(F=35.700,P<0.001),which was greater in the BLM and BLM+Bai than in the control group,and lower in the BLM+Bai than in the BLM group(P<0.05).The relative expression of pAMPK and NRF2 was 0.28±0.02 and 0.38±0.09 after Compound C+Baicalein treatment,which was lower than that of 0.56±0.07 and 0.60±0.08 in the BLM+Bai group(P<0.05).The relative expression of ROS increased from 1.75±0.16 to 3.45±0.12(P<0.001).The proportion of positivity in the BLM+Bai+Compound C,BLM+Bai and control groups was 47.30%±4.29%,18.18%±3.89%and 7.70%±3.53%(F=109.700,P<0.001),respectively,with the BLM+Bai+Compound C group being higher than that in the BLM+Bai group(P<0.05).Conclusion The downregulation of Sirt1 expression in patients with osteoarthritis leads to FLS senescence and accelerates the progression of osteoarthritis.Baicalein can inhibit FLS senescence by activating Sirt1/AMPK/NRF2 pathway,which may delay the progression of osteoarthritis and improve the function of chondrocytes.