PRP通过NRF2/HO-1通路对LPS诱导的BV2小胶质细胞神经炎症的保护作用

Protective effect of platelet-rich plasma on LPS-induced neuroinflammation in BV2 microglia through NRF2/HO-1 pathway

目的 探究富血小板血浆(platelet-rich plasma, PRP)对脂多糖(lipopolysaccharide, LPS)诱导的BV2细胞炎症反应保护作用与机制。方法 BV2小胶质细胞分成正常对照组、10%PRP对照组、LPS组(LPS诱导)、3%PRP+LPS组(LPS诱导,3%PRP预处理)、5%PRP+LPS组(LPS诱导,5%PRP预处理)和10%PRP+LPS组(LPS诱导,10%PRP预处理),CCK-8测定BV2细胞增殖。共聚焦显微镜测定BV2细胞线粒体膜电位水平,荧光法检测ROS生成情况,Griess法测定NO水平。Western blot检测IL-6、TNF-α、BACH1、GPX4、NRF2和HO-1蛋白表达。此外,用HO-1抑制剂处理BV2小胶质细胞后实验分为正常对照组,LPS组,ZnPP+LPS组,10%PRP+LPS组,ZnPP+LPS+10%PRP,Western blot检测HO-1、IL-6和TNF-α蛋白表达。结果 与正常对照组相比,PRP促进BV2细胞增殖(P<0.01);LPS组线粒体膜电位下降,ROS生成增多,NO、IL-6、TNF-α、BACH1水平升高(P<0.01),而GPX4、NRF2、HO-1表达水平下降(P<0.01)。与LPS组相比,3%PRP+LPS组、5%PRP+LPS组、10%PRP+LPS组BV2细胞增殖活性升高,线粒体膜电位升高;ROS、NO、IL-6、TNF-α、BACH1水平降低(P<0.01);GPX4、NRF2、HO-1在不同浓度PRP(3%、5%、10%)组表达升高(P<0.01)。另外,使用HO-1抑制剂结果显示,与LPS组比较,ZnPP+LPS组IL-6和TNF-α表达增高;与10%PRP+LPS+ZnPP组比较,HO-1抑制剂能够逆转PRP对LPS诱导下BV2细胞IL-6和TNF-α表达的影响(P<0.01)。结论 PRP可以通过激活NRF2/HO-1信号通路来抑制LPS诱导的BV2小胶质细胞炎症反应。

Objective To investigate the protective effect and mechanism of platelet-rich plasma(PRP) on lipopolysaccharide(LPS)-induced inflammatory response in BV2 cells. Methods BV2 microglia were divided into normal control group, 10%PRP control group, LPS group(LPS induction), 3%PRP+LPS group(LPS induction, 3%PRP pretreatment), 5%PRP+LPS group(LPS induction, 5%PRP pretreatment), 10%PRP+LPS group(LPS induction, 10%PRP pretreatment), and the proliferation of BV2 cells was measured by CCK-8. The mitochondrial membrane potential of BV2 cells was measured by confocal microscopy, ROS was measured by fluorescence method, and NO was measured by Griess method. The protein expressions of IL-6, TNF-α, BACH1, GPX4, NRF2 and HO-1 were detected by Western blot. In addition, BV2 microglia were treated with HO-1 inhibitor and divided into normal control group, LPS group, ZnPP+LPS group, 10%PRP+LPS group, ZnPP+LPS+10%PRP group, and the protein expressions of HO-1, IL-6 and TNF-α were detected by Western blot. Results Compared with normal control group, PRP promoted the proliferation of BV2 cells(P<0.01). The mitochondrial membrane potential decreased, ROS production increased, the levels of NO, IL-6, TNF-α and BACH1 increased(P<0.01). However, the expression levels of GPX4, NRF2 and HO-1 decreased(P<0.01) in LPS group. Compared with LPS group, the proliferation activity and mitochondrial membrane potential of BV2 cells in 3%PRP+LPS, 5%PRP+LPS and 10%PRP+LPS groups significantly increased. The levels of ROS, NO, IL-6, TNF-α and BACH1 significantly decreased(P<0.01). The expressions of GPX4, NRF2 and HO-1 in different concentrations of PRP(3%, 5% and 10%) increased(P<0.01). Moreover, the expression of IL-6 and TNF-α in ZnPP+LPS group was significantly higher than that in LPS group after HO-1 inhibitor treatment. Compared with 10%PRP+LPS+ZnPP group, HO-1 inhibitor could reverse the effect of PRP on the expression of IL-6 and TNF-α in LPS-induced BV2 cells(P<0.01). Conclusion PRP inhibits the inflammatory response of BV2 microglia induced by LPS by activating the NRF2/HO-1 signaling pathway.