基于p38MAPK通路探讨疏肝健脾方药含药血清对LPS刺激下大鼠Kupffer细胞炎症损伤保护作用的机制

Based on p38MAPK pathway investigate the effects of soothing liver and invigorating spleen recipes contained serum on LPS-stimulated inflammatory injury in Kupffer cells of rats

目的基于p38MAPK通路探讨疏肝健脾方药含药血清对脂多糖(LPS)刺激大鼠Kupffer细胞炎症损伤的保护作用。方法体外培养并鉴定SD大鼠Kupffer细胞,提取并制备疏肝健脾方药含药血清,在LPS刺激大鼠Kupffer细胞产生炎症反应基础上,对Kupffer细胞进行疏肝健脾方药含药血清、p38MAPK抑制剂药物干预,ELISA法检测Kupffer细胞上清液中白介素(IL)-6、肿瘤坏死因子(TNF)-α含量的表达,Western印迹检测Kupffer细胞TLR4、p38MAPK、p-p38MAPK蛋白的表达。结果疏肝健脾方药含药血清提取量约为每只2~3 ml,大鼠获得纯化并经免疫荧光法鉴定Kupffer细胞(0.5~1.0)×107个,Typan blue染色测定细胞活力均在95%以上;LPS组较空白血清组IL-6、TNF-α水平均有显著升高(P<0.01);与LPS组比较,各药物干预组IL-6、TNF-α表达水平显著降低(P<0.01);与空白血清组比较,LPS组的p38MAPK、p-p38MAPK、TLR4蛋白表达均有显著升高(P<0.01);与LPS组比较,SB239063能显著下调p38MAPK的蛋白表达水平(P<0.01),肝健脾组亦有下调,但无显著差异(P>0.05);疏肝健脾组、SB239063组的p-p38MAPK蛋白表达水平均下调显著(P<0.01);TLR4蛋白表达水平以疏肝健脾组下调具有显著性差异(P<0.01),SB239063组下调无显著差异(P>0.05)。结论疏肝健脾方药可能对LPS刺激大鼠Kupffer细胞炎症损伤发挥保护作用,而含药血清可能是其重要作用途径之一。

Objective To investigate the protective mechanism of the soothing liver and invigorating spleen recipes contained serum on the Kupffer cells( KCs) of the inflammatory injured rats based on p38 mitogen activated protein kinases( p38MAPK) pathway. Methods Thirty SD rats were randomly divided into normal control,soothing liver and invigoration spleen recipes treatment( SGJP) and KCs extraction groups. After isolation,cultivating and identifying KCs of SD rats,at the same time,soothing liver and invigorating spleen recipes contained serum was made up. KCs of rats with inflammation under LPS stimulation were interfered by normal serum,soothing liver and invigorating spleen recipes contained serum and p38 MAPK inhibitor drug( SB239063) respectively. The expressions of interleukin( IL)-6 and tumor necrosis factor-alpha( TNF-α) levels on KCs supernatant were tested by ELISA,while the expressions of KCs toll-like receptor( TLR) 4,p38 MAPK and P-P38 mitogen-activated protein kinase( p-p38MAPK) were detected by Western blot. Results The extracting volume of soothing liver and invigorating spleen recipes contained serum of each rats was about 2 ~ 3 ml( per rats). Purifying and identifying KCs through immunofluorescence assay,( 0. 5 ~ 1. 0) ×107units of KCs were ascertained. Both IL-6 and TNF-α levels in LPS group were significantly elevated more than those of normal serum group( P<0. 01). Compared with those of LPS group,IL-6 and TNF-α expression levels of the drug intervention group were significantly decreased( P<0. 01). Compared with those of normal serum group,protein expressions of TLR4,p38 MAPK,p-p38 MAPK in LPS group were significantly increased( P<0. 01). Compared with that of LPS group,TLR4 protein expression level in SGJP group had a significant difference( P<0. 01). The p-p38 MAPK protein expression level of soothing liver and invigorating spleen recipes contained serum and SB239063 groups were significantly down-regulated( P < 0. 01). Conclusions Soothing liver and invigorating spleen recipes contained serum might have protective effects on the LPS-stimulated inflammatory injury in KCs of rats and contained serum might be one of important pathways of soothing liver and invigorating spleen recipes treating NASH.