Shi Jianfeng, Zhao Weibo, Ying Haijian, Zhang Ying, Du Juping, Chen Shuaishuai, Li Jun, Shen Bo
Department of Clinical Laboratory, Taizhou Hospital of Zhejiang Province, Affiliated Hospital of Wenzhou Medical College, Taizhou, Zhejiang Province, China.
Department of Orthopedics, Taizhou Hospital of Zhejiang Province, Affiliated Hospital of Wenzhou Medical University, Taizhou, Zhejiang Province, China.
Int J Rheum Dis. 2018 Nov;21(11):2002-2010. doi: 10.1111/1756-185X.13198. Epub 2017 Nov 3.
Nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome is known for activating pro-inflammatory cytokines in knee osteoarthritis (OA). This study was performed to identify whether NLRP3 inflammasome can be triggered by lipopolysaccharides (LPS) and adenosine triphos adenine (ATP), which are positively related with knee OA severity in joint-spaces, in human fibroblast-like synoviocytes (FLS), and to identify whether estrogen would inhibit the activation of NLRP3 inflammasome.
In the present study, human FLS were isolated from the knee OA in patients during arthroplasty, and were treated with LPS and ATP in the presence or absence of estradiol (E2). The messenger RNA (mRNA) and protein levels of NLRP3 components were analyzed by real-time polymerase chain reaction and western blotting, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to examine interleukin (IL)-1β and IL-18 content in the supernatant. Estrogen receptor α inhibitor MPP and β inhibitor PHTPP were used to explore how E2 works.
Our results demonstrated that treatment with LPS and ATP increased significantly both in mRNA and protein levels of all the NLRP3 inflammasome components, and triggered the NLRP3 inflammasome, followed by upregulated IL-1β and IL-18 in the cell supernatant. E2 appeared to inhibit the activation of NLRP3 inflammasome by diminishing mRNA and protein levels of NLRP3 through estrogen receptor β, and decreased the expression of IL-1β and IL-18 as well.
These results suggested the increased LPS and ATP in joint-space may promote knee OA by NLRP3 inflammasome and E2 may exert a protective effect by inhibiting the activation of NLRP3 inflammasome in FLS.
核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性小体以激活膝骨关节炎（OA）中的促炎细胞因子而闻名。本研究旨在确定在人成纤维样滑膜细胞（FLS）中，与膝OA严重程度呈正相关的脂多糖（LPS）和三磷酸腺苷（ATP）是否能触发NLRP3炎性小体，以及雌激素是否会抑制NLRP3炎性小体的激活。
在本研究中，从行关节置换术的膝OA患者中分离出人FLS，并在有或没有雌二醇（E2）的情况下用LPS和ATP进行处理。分别通过实时聚合酶链反应和蛋白质印迹分析NLRP3组分的信使核糖核酸（mRNA）和蛋白质水平。采用酶联免疫吸附测定（ELISA）检测上清液中白细胞介素（IL）-1β和IL-18的含量。使用雌激素受体α抑制剂MPP和β抑制剂PHTPP来探究E2的作用机制。
我们的结果表明，用LPS和ATP处理后，所有NLRP3炎性小体组分的mRNA和蛋白质水平均显著升高，并触发了NLRP3炎性小体，随后细胞上清液中的IL-1β和IL-18上调。E2似乎通过雌激素受体β降低NLRP3的mRNA和蛋白质水平来抑制NLRP3炎性小体的激活，同时也降低了IL-1β和IL-18的表达。
这些结果表明，关节腔中LPS和ATP的增加可能通过NLRP3炎性小体促进膝OA，而E2可能通过抑制FLS中NLRP3炎性小体的激活发挥保护作用。
