富血小板血浆对脂多糖诱导的星形胶质细胞炎性反应的作用

Effects of platelet-rich plasma on lipopolysaccharide-induced astrocytic inflammation

目的了解富血小板血浆(PRP)对脂多糖诱导(LPS)的星形胶质细胞活化、增殖及炎症相关因子释放的影响。方法无菌条件下取10只出生1-3d SD大鼠乳鼠,处死并取大脑皮质,使用胰酶消化大脑皮质组织,清洗、离心并过滤后得到原代大鼠星形胶质细胞。使用胶质纤维酸性蛋白(GFAP)免疫荧光染色鉴定所得细胞是否为大鼠星形胶质细胞。建立LPS诱导的星形胶质细胞炎症体外模型,分为5组:空白对照(BC)及LPS、LPS+2.5% PRP、LPS+5% PRP以及LPS+10% PRP组,各LPS组均以终浓度为1μg/mL LPS预处理24 h后,加不同浓度PRP处理24 h。提取各组大鼠星形胶质细胞细胞蛋白,以WB法检测大鼠星形胶质细胞GFAP蛋白含量、CCK-8法检测细胞存活率,Griess法检测细胞NO释放量,ELISA法检测细胞炎症因子TNF-α和IL-6表达量。结果1)GFAP(抗体)标记的阳性细胞数>95%,说明所培养的细胞为SD大鼠星形胶质细胞。2)5组GFAP表达水平(灰度值):LPS组(3.49±0.15)较BC组(1.23±0.18)明显增加(P<0.05),而3个LPS+(2.5%、5%、10%)PRP组(2.77±0.32、1.83±0.14、1.78±0.24)较LPS组明显降低(P<0.05)。3)细胞增殖率(%):LPS(组)诱导12、24、48 h(125.47±5.4、144.72±6.5、151.49±7.1)后较BC组(100±6.2、100±5.2、100±8.0)明显上升(P<0.05);而加入2.5%、5%、10%PRP处理12 h(113.04±7.4、94.41±4.2、90.06±2.1)、24 h(108.21±7.9、93.44±7.4、83.59±4.1)和48 h(98.02±9.3、78.22±9.4、70.1±8.4)后较LPS组明显下降(P<0.05)。4)5组细胞上清液中的NO含量(μmol/L):LPS组(13.28±0.58)较BC组(3.01±0.15)明显上升(P<0.05);而加入2.5%、5%、10%PRP(组)处理(9.98±0.72、7.19±0.91、6.82±0.46)后较LPS组明显降低(P<0.05)。5)TNF-α和IL-6表达(ng/L):LPS(组)处理后(TNF-α为4.204±0.04、IL-6为0.483±0.02)较BC组(TNF-α为1.763±0.05、IL-6为0.296±0.02)均明显上升(P<0.05);而加入2.5%、5%、10%PRP(组)处理后(TNF-α为3.234±0.09、3.496±0.05、3.260±0.17,IL-6为0.403±0.01、0.381±0.01、0.386±0.02)较LPS组均明显下降(P<0.05)。结论PRP能够抑制大鼠模型LPS诱导的星形胶质细胞活化和增殖,有效改善大鼠星形胶质细胞炎症损伤。

Objective To investigate the effects of platelet-rich plasma(PRP)on the activation,proliferation and release of inflammatory related factors of lipopolysaccharide(LPS)-induced astrocytes.Methods 10 neonatal SD rats were scarified on germfree conditions and the cerebral cortex was taken before digested with pancreatin.After washing and centrifuging,the primary rat astrocytes were obtained.Glial fibrillary acidic protein(GFAP)immunofluorescence staining was used to identify rat astrocytes.An in vitro model of LPS-induced inflammation in astrocyte was established,which was divided into 5 groups:blank control(BC),LPS,LPS+2.5%PRP,LPS+5%PRP and LPS+10% PRP group.Each LPS group were treated with a final concentration of 1μg/mL LPS for 24h,and then different concentrations of PRP were added for another 24 h.The GFAP protein content of the rat astrocytes was detected by Western blot,and the cell viability was detected by the CCK-8 method.The release of NO was detected by the Griess method,and the expression of TNF-1 and IL-6 in the cell was detected by ELISA.Results 1)the number of positive cells labeled was>95%,indicating that the cultured cells were rat astrocytes.2)The expression of GFAP(gray value):LPS group(3.49±0.15)was significantly higher than that in BC group(1.23±0.18)(P<0.05),while three LPS+PRP groups(2.77±0.32,1.83±0.14,1.78±0.24)decreased significantly than that of LPS group(P<0.05).3)The cell proliferation rate(%):LPS(group)induced for 12,24 and 48h was 125.47±5.4,144.72±6.5,and 151.49±7.1,respectively,and the cell proliferation rate of LPS groups were significantly higher than BC groups(100±6.2,100±5.2,and 100±8.0)(P<0.05),and then different concentrations of PRP groups were added for 12 h(113.04±7.4,94.41±4.2,and 90.06±2.1),24h(108.21±7.9,93.44±7.4,and 83.59±4.1)or 48h(98.02±9.3,78.22±9.4 and 70.1±8.4),the cell proliferation rate of PRP groups were significantly decreased compared with LPS group(P<0.05).4)The NO content in the supernatant of astrocytes(μmol/L):the NO content of the LPS group(13.28±0.58)was significantly higher than that in BC group(3.01±0.15)(P<0.05);and then LPS+different concentrations of PRP groups decreased significantly(P<0.05).5)TNF-α and IL-6 expression(ng/L):the TNF-α(4.204±0.04)and IL-6 expression(0.483±0.02)of LPS groups increased significantly compared with that in BC groups(1.763±0.05 and 0.296±0.02)(P<0.05);and LPS+PRP groups(TNF-α:3.234±0.09,3.496±0.05,and 3.260±0.17;IL-6:0.403±0.01,0.381±0.01,and 0.386±0.02)were significantly lower than that of LPS groups(P<0.05).Conclusion PRP can significantly inhibit the activation and proliferation of astrocytes induced by LPS which can improve the inflammatory injury of rat astrocytes.