背景:脂肪组织来源的干细胞(ADSC)移植已被证明可有效治疗严重的肝脏疾病。ADSC的预激活增强了它们的治疗功效。然而,这些影响尚未与胆汁淤积性肝损伤相关。
方法:在本研究中,采用胆管结扎法(BDL)建立C57BL/6小鼠胆汁淤积性肝损伤模型。通过尾静脉注射给小鼠施用具有或不具有肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)预处理的人ADSCs(hADSC)。通过组织学染色评估hADSCs对BDL诱导的肝损伤的疗效,实时定量PCR(RT-qPCR),蛋白质印迹,和酶联免疫吸附测定(ELISA)。体外,研究了hADSC条件培养基对肝星状细胞(HSC)活化的影响。小干扰RNA(siRNA)用于敲除hADSC中的环氧合酶-2(COX-2)。
结果:TNF-α/IL-1β预处理可以下调免疫原性基因表达,提高hADSC的移植效率。与对照hADSCs(C-hADSCs)相比,TNF-α/IL-1β预处理的hADSCs(P-hADSCs)可明显减轻BDL诱导的肝损伤,正如肝细胞死亡减少所证明的那样,Ly6G+中性粒细胞浸润减弱,促炎细胞因子TNF-α的表达降低,IL-1β,C-X-C基序趋化因子配体1(CXCL1),和C-X-C基序趋化因子配体2(CXCL2)。此外,P-hADSC显著延缓BDL诱导的肝纤维化的发展。体外,与来自C-hADSC的条件培养基相比,来自P-hADSC的条件培养基显著抑制HSC活化。机械上,TNF-α/IL-1β上调COX-2表达并增加前列腺素E2(PGE2)分泌。通过siRNA转染阻断COX-2逆转了P-hADSC产生PGE2的益处,HSC激活,和肝纤维化进展。
结论:结论:我们的结果表明,TNF-α/IL-1β预处理可增强hADSCs对胆汁淤积性肝损伤小鼠的疗效,部分通过COX-2/PGE2途径。
Adipose tissue-derived stem cell (ADSC) transplantation has been shown to be effective for the management of severe liver disorders. Preactivation of ADSCs enhanced their therapeutic efficacy. However, these effects have not yet been examined in relation to cholestatic liver injury.
In the present study, a cholestatic liver injury model was established by bile duct ligation (BDL) in male C57BL/6 mice. Human ADSCs (hADSCs) with or without tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) pretreatment were administrated into the mice via tail vein injections. The efficacy of hADSCs on BDL-induced liver injury was assessed by histological staining, real-time quantitative PCR (RT-qPCR), Western blot, and enzyme-linked immune sorbent assay (ELISA). In vitro, the effects of hADSC conditioned medium on the activation of hepatic stellate cells (HSCs) were investigated. Small interfering RNA (siRNA) was used to knock down cyclooxygenase-2 (COX-2) in hADSCs.
TNF-α/IL-1β preconditioning could downregulate immunogenic gene expression and enhance the engraftment efficiency of hADSCs. Compared to control hADSCs (C-hADSCs), TNF-α/IL-1β-pretreated hADSCs (P-hADSCs) significantly alleviated BDL-induced liver injury, as demonstrated by reduced hepatic cell death, attenuated infiltration of Ly6G + neutrophils, and decreased expression of pro-inflammatory cytokines TNF-α, IL-1β, C-X-C motif chemokine ligand 1 (CXCL1), and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, P-hADSCs significantly delayed the development of BDL-induced liver fibrosis. In vitro, conditioned medium from P-hADSCs significantly inhibited HSC activation compared to that from C-hADSCs. Mechanistically, TNF-α/IL-1β upregulated COX-2 expression and increased prostaglandin E2 (PGE2) secretion. The blockage of COX-2 by siRNA transfection reversed the benefits of P-hADSCs for PGE2 production, HSC activation, and liver fibrosis progression.
In conclusion, our results suggest that TNF-α/IL-1β pretreatment enhances the efficacy of hADSCs in mice with cholestatic liver injury, partially through the COX-2/PGE2 pathway.