PDF(Pubmed)
Abstract：
UNASSIGNED: Intervertebral disc (IVD) degeneration is associated with chronic back pain. We previously demonstrated that the phosphatase pleckstrin homology domain and leucine-rich repeat protein phosphatase (PHLPP) 1 was positively correlated with IVD degeneration and its deficiency decelerated IVD degeneration in both mouse IVDs and human nucleus pulposus (NP) cells. Small molecule PHLPP inhibitors may offer a translatable method to alleviate IVD degeneration. In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype.
UNASSIGNED: Tail IVDs of 5-month-old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot.
UNASSIGNED: In a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586-induced regenerative potential, especially in males.
UNASSIGNED: Our study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration.
UNASSIGNED: Tail IVDs of 5-month-old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot.
UNASSIGNED: In a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586-induced regenerative potential, especially in males.
UNASSIGNED: Our study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration.
摘要：
■椎间盘（IVD）变性与慢性背痛有关。我们先前证明了磷酸酶pleckstrin同源域和富含亮氨酸的重复蛋白磷酸酶（PHLPP）1与IVD变性呈正相关，其缺乏会减缓小鼠IVD和人髓核（NP）细胞中的IVD变性。小分子PHLPP抑制剂可提供减轻IVD变性的可翻译方法。在这项研究中,我们测试了两种PHLPP抑制剂NSC117079和NSC45586在促进健康NP表型方面的有效性。
■收集5个月大的野生型小鼠的尾IVD,并在离体低血清条件下用NSC117079或NSC45586处理。进行苏木精和伊红染色以检查IVD结构和NP细胞形态。通过免疫组织化学分析KRT19的表达。通过TUNEL测定评估细胞凋亡。从患有IVD变性的患者获得人NP细胞。KRT19、ACAN、SOX9和MMP13通过实时qPCR分析，并通过免疫印迹分析FOXO1的AKT磷酸化和蛋白表达。
■在小鼠IVD器官培养模型中，NSC45586，而非NSC117079，保留了空泡状的脊索细胞形态和KRT19表达，同时抑制了细胞凋亡，抵消血清剥夺引起的退行性变化，尤其是男性。同样，在退化的人类NP细胞中，NSC45586增加了细胞活力和KRT19，ACAN，和SOX9并降低MMP13的表达,而NSC117079处理仅增加KRT19的表达。机械上,NSC45586处理增加NP细胞中FOXO1蛋白的表达，并抑制FOXO1抵消NSC45586诱导的再生潜力，尤其是男性。
■我们的研究表明NSC45586在促进NP细胞健康方面是有效的，尤其是男性，提示PHLPP在NP细胞稳态中起关键作用,NSC45586可能是治疗IVD变性的潜在候选药物。
■收集5个月大的野生型小鼠的尾IVD,并在离体低血清条件下用NSC117079或NSC45586处理。进行苏木精和伊红染色以检查IVD结构和NP细胞形态。通过免疫组织化学分析KRT19的表达。通过TUNEL测定评估细胞凋亡。从患有IVD变性的患者获得人NP细胞。KRT19、ACAN、SOX9和MMP13通过实时qPCR分析，并通过免疫印迹分析FOXO1的AKT磷酸化和蛋白表达。
■在小鼠IVD器官培养模型中，NSC45586，而非NSC117079，保留了空泡状的脊索细胞形态和KRT19表达，同时抑制了细胞凋亡，抵消血清剥夺引起的退行性变化，尤其是男性。同样，在退化的人类NP细胞中，NSC45586增加了细胞活力和KRT19，ACAN，和SOX9并降低MMP13的表达,而NSC117079处理仅增加KRT19的表达。机械上,NSC45586处理增加NP细胞中FOXO1蛋白的表达，并抑制FOXO1抵消NSC45586诱导的再生潜力，尤其是男性。
■我们的研究表明NSC45586在促进NP细胞健康方面是有效的，尤其是男性，提示PHLPP在NP细胞稳态中起关键作用,NSC45586可能是治疗IVD变性的潜在候选药物。
