Supplementary Material for: Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway

Background/Aims: Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy Methods: We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in in vivo studies. Results: The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our in vitro findings were supported by our in vivo evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. Conclusion: LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy.

研究背景与目的：肥厚性黄韧带（hypertrophic ligamentum flavum, LF）是引发腰椎管狭窄症的主要病因。本团队前期研究发现，溶血磷脂酸（lysophosphatidic acid, LPA）的高表达与LF肥厚呈正相关。本研究旨在进一步阐明LPA调控LF肥厚的具体机制。
方法：本研究通过聚合酶链式反应（PCR）与蛋白质印迹法（western blotting）检测人LF细胞中溶血磷脂酸受体1（LPAR1）的表达水平；分别构建LPAR1敲低与过表达的LF细胞系，检测细胞活力、细胞周期、凋亡率及相关分子机制；并通过人体样本与体内实验验证LF肥厚现象及其分子机制。
结果：相较于健康对照组，肥厚性LF组织与细胞中LPA及其受体LPAR1的表达水平显著升高。进一步研究发现，LPA可通过结合LPAR1促进LF细胞增殖；LPAR1的敲低或过表达可改变LPA对LF细胞增殖的调控作用，进一步验证了上述结论。此外，LPA还可通过LPAR1抑制LF细胞凋亡。重要的是，本研究证实LPA与LPAR1结合后可启动AKT磷酸化，进而调控细胞增殖与凋亡。体外实验结果得到了体内实验的支持：冻干LPA可通过LPAR1-AKT信号通路显著诱导LF肥厚。更为关键的是，通过凝胶海绵植入递送Ki16425靶向抑制LPAR1，可有效减轻LPA介导的LF肥厚。综上，本研究数据表明，LPA通过结合LPAR1激活AKT信号级联反应，从而诱导LF细胞增殖并抑制细胞凋亡；采用Ki16425靶向调控该信号通路，有望成为预防LF肥厚的潜在治疗策略。
结论：LPA-LPAR1-AKT信号轴的激活与LF细胞的增殖及存活呈正相关。LPAR1可作为新型药物靶点，为治疗LF肥厚的新型治疗方法开发提供新方向。