Changli Zhang, Madeleine D. Gordon, Katherine M. Joseph, Martha E. Diaz-Hernandez, Hicham Drissi, Svenja Illien-Jünger
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In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>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.</p>\n </section>\n </div>","PeriodicalId":14876,"journal":{"name":"JOR Spine","volume":"7 1","pages":""},"PeriodicalIF":3.4000,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jsp2.1306","citationCount":"0","resultStr":"{\"title\":\"Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health\",\"authors\":\"Changli Zhang, Madeleine D. 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引用次数: 0
摘要
椎间盘(IVD)退变与慢性背痛有关。我们之前已经证明，磷酸酶pleckstrin同源结构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，并且在小鼠IVD和人髓核(NP)细胞中，它的缺乏减缓了IVD变性。小分子PHLPP抑制剂可能提供一种可翻译的方法来减轻IVD变性。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月龄野生型小鼠的尾ivd，在低血清条件下用NSC117079或NSC45586体外处理。苏木精和伊红染色检测IVD结构和NP细胞形态。免疫组化分析KRT19的表达。TUNEL法检测细胞凋亡。从IVD变性患者中获得人NP细胞。real - time qPCR检测KRT19、ACAN、SOX9和MMP13基因表达，免疫印迹检测AKT磷酸化和FOXO1蛋白表达。在小鼠IVD器官培养模型中，NSC45586(而非NSC117079)在抑制细胞凋亡的同时，保留了空泡化脊索细胞形态和KRT19表达，抵消了血清剥夺引起的退行性改变，尤其是在雄性小鼠中。同样，在退化的人NP细胞中，NSC45586增加了细胞活力和KRT19、ACAN和SOX9的表达，降低了MMP13的表达，而NSC117079只增加了KRT19的表达。在机制上，NSC45586处理增加了NP细胞中FOXO1蛋白的表达，抑制FOXO1抵消了NSC45586诱导的再生潜能，尤其是在雄性中。我们的研究表明，NSC45586能有效促进NP细胞健康，特别是在男性中，这表明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。
Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health
Background
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.
Methods
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.
Results
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.
Conclusions
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.
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