磷肌酰脂醇特异性磷脂酶C对内毒素介导枯否细胞激活的抑制作用

Inhibitory role and mechanism of phosphatidylinositol-specific phospholipase C in Kupffer cells activation induced by endotoxin

目的研究磷肌酰脂醇特异性磷脂酶C(PI-PLC)对内毒素介导的肝Kupffer细胞(枯否细胞,KCs)激活及其CD14表达的抑制作用。方法Wistar大鼠20只,分为PI-PLC组和LPS组,测定细胞培养液中TNF-α和IL-6含量变化,用免疫组化观察KCs膜CD14和核NF-κB的相对活性,测定KCs中CD14、TNF-α和IL-6的mRNA表达和KCs膜CD14蛋白含量变化。结果不同浓度的LPS刺激60 min后,PI-PLC组培养液中TNF-α与IL-6的含量随LPS浓度的增高而增加,但明显低于LPS组(P<0.01);CD14抗体染色显示,PI-PLC组部分KCs为弱阳性,而LPS组KCs为阳性细胞;NF-κB P65抗体染色显示,PI-PLC组部分KCs为弱阳性细胞,而LPS组KCs为强阳性细胞;PI-PLC组NF-κB活性在10μg/mL以上LPS刺激下才有升高,其相对光密度值显著低于LPS组(P<0.01);在相同浓度LPS刺激后,PI-PLC组CD14、TNF-α和IL-6的mRNA表达显著低于LPS组(P<0.01);PI-PLC组在相同浓度LPS刺激120 min后CD14蛋白表达才明显,LPS组在100μg/mL LPS刺激后30 min CD14蛋白开始升高,两者有显著性差异(P<0.01)。结论PI-PLC对LPS介导的KCs激活有明显的抑制作用,其机制可能与抑制KCs中CD14蛋白的表达有关。

Objective To study the inhibitory role of phosphatidylinositol- specific phosphohpase C（PI - PIE） in liver Kupffer cells （KCs） activation and its CD14 expression induced by endotoxin.Methods KCs were isolated from the Wistar rat liver by the in situ collagenase perfusion technique. The isolated KCs were divided randomly into two groups：LPS group and PI - PIE group. The culture supematants of these cells were then collected for treasuring the levels of tumor necrosis factor（TNF） - α and interleukin（IL） - 6. CD14 protein were observed on KCs surface with immunecytechemical analysis. The activation of NF- κB in KCs was revealed with electrophorefic mobility shift assay（EMSA）. The expression of CD14, TNF - α and IL- 6 mRNA in KCs were detected by reversal transcription - polymerase chain reaction（RT - PCR）. Expression of CD14 protein on the surface of KCs was detected by Western blotting analysis.Results When the level of culture supernatant LPS elevated, the level of culture supernatant TNF - α and IL - 6 in PI - PLC group were increased at 1 h after the stimulation of LPS, but were lower markedly compared with those in LPS group（ P 〈 0.01） ; Parts of KCs in PI - PIE group showed weak positive by using anti - CD14 antibody staining, but the KCs in LPS group were all positive; Parts of KCs in PI - PIE group showed weak positive by using anti - NF- κ BP^65 antibody staining, but the KCs in LPS group all showed strong positive; The activation of NF- κB in KCs of PI - PIE group was elevated only when the concentration of LPS was above 10μg/mL, and its OD was lower markedly compared with that in LPS group（ P 〈 0.01） ; Under the stimulation of LPS with the same concentration, the expression of CD14mRNA, TNF- α mRNA and IL- 6mRNA were lower markedly compared with those in LPS group（ P 〈 0.01） ; The expression of CD14 protein in LPS group increased 30 minutes after the stimulation of LPS（100μg/mL）, but in PI- PIE group, it was 120 mintues after the same LPS stimulation when the expression of CD14 protein were obvious. There was significant differences between PI - PIE group and LPS group（ P 〈 0.01 ）. Conclusion PI - PIE has obviously inhibitory role in LPS - mediated KCs activation, which may be associated with the mechanism by inhibiting the expression of CD14 protein in KCs.