背景:骨骼肌的脂肪浸润已被认为是许多退行性肌肉疾病的共同特征。视黄醇结合蛋白4(RBP4)是一种脂肪因子,已被证明与老年人肌肉减少症的存在和严重程度有关。然而,RBP4在肌肉萎缩中的确切作用和潜在机制尚不清楚.
方法:在野生型和RBP4基因敲除小鼠中构建去神经诱导的肌肉萎缩模型。为了改变RBP4的表达,小鼠肌肉注射无视黄醇的RBP4(apo-RBP4),视黄醇结合RBP4(holo-RBP4)或口服灌胃RBP4抑制剂A1120。用siRNA或靶向视黄醇6(STRA6)/Janus激酶2(JAK2)/信号转导子和转录激活因子3(STAT3)途径的信号传导受体和转运蛋白的特异性抑制剂处理全RBP4刺激的C2C12肌管。脂肪积累,肌纤维横截面积,分析了肌管直径以及肌肉萎缩标志物和肌生成标志物的表达。
结果:骨骼肌中RBP4的表达水平从7天开始显着上调2倍以上,并在去神经支配后持续28天。免疫荧光分析表明,增加的RBP4位于失神经骨骼肌的浸润脂肪区域。RBP4基因敲除减轻了神经支配诱导的脂肪浸润和肌肉萎缩,同时减少了萎缩标志物Atrogin-1和MuRF1的表达,并增加了肌生成调节剂MyoD和MyoG的表达。相比之下,注射视黄醇结合的holo-RBP4聚集的去神经支配诱导的异位脂肪积累和肌肉萎缩。始终如一,holo-RBP4刺激对C2C12肌管直径和肌纤维横截面积的减小也具有剂量依赖性作用,以及Atrogin-1和MuRF1表达的增加和MyoD和MyoG表达的减少。机械上,holo-RBP4处理增加了其膜受体STRA6的表达(>3倍),并促进了下游JAK2和STAT3的磷酸化。通过特异性siRNA或抑制剂抑制STRA6/JAK2/STAT3途径可以降低Atrogin-1和MuRF1的表达(>50%),并降低全RBP4处理的C2C12肌管中MyoD和MyoG的表达(>3倍)。RBP4特异性药理拮抗剂A1120显著抑制STRA6/JAK2/STAT3通路的激活,改善异位脂肪浸润,并保护小鼠免受神经支配诱导的肌肉萎缩(肌纤维横截面积增加30%)。
结论:结论:我们的数据显示,RBP4通过STRA6依赖性和JAK2/STAT3通路介导的去神经骨骼肌机制促进脂肪浸润和肌肉萎缩.我们的结果表明,降低RBP4水平可能是预防和治疗肌肉萎缩的有希望的治疗方法。
BACKGROUND: Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear.
METHODS: Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (
STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed.
RESULTS: The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7 days and sustained for 28 days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor
STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of
STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of
STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice.
CONCLUSIONS: In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a
STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.