Klotho-mediated activation of the anti-oxidant Nrf2/ARE signal pathway affects cell apoptosis, senescence and mobility in hypoxic human trophoblasts: involvement of Klotho in the pathogenesis of preeclampsia

Abstract

The anti-aging gene Klotho is implicated in the pathogenesis of preeclampsia (PE), which is a pregnancy disease characterized by hypertension and proteinuria. Oxidative stress is closely associated with the worse outcomes in PE, and Klotho can eliminate Reactive Oxygen Species (ROS), but it is still unclear whether Klotho regulates PE pathogenesis through modulating oxidative damages. Here, by analyzing the clinical data, we found that Klotho was aberrantly downregulated in PE umbilical cord serum and placental tissues, compared to their normal counterparts. In in vitro experiments, the human trophoblasts were subjected to hypoxic pressure to establish the PE models, and we confirmed that hypoxia also decreased the expression levels of Klotho in those trophoblasts. In addition, through performing functional experiments, we confirmed that hypoxia promoted oxidative damages, cell apoptosis and senescence, whereas suppressed cell invasion in human trophoblasts, which were all reversed overexpressing Klotho. The following mechanical experiments verified that Klotho increased the levels of nuclear Nrf2, total Nrf2, SOD2 and NQO1 to activate the anti-oxidant Nrf2/ARE signal pathway, and silencing of Nrf2 abrogated the protective effects of Klotho overexpression on hypoxic human trophoblasts. Consistently, in in vivo experiments, Klotho overexpression restrained oxidative damages and facilitated cell mitosis in PE rats' placental tissues. In conclusion, this study validated that Klotho activated the Nrf2/ARE signal pathway to eliminate hypoxia-induced oxidative damages, cell apoptosis and senescence to recover normal cellular functions in human trophoblasts, and our data supported that Klotho could be used as novel biomarker for PE diagnosis and treatment.

Keywords: Human trophoblasts; Hypoxia; Nrf2/ARE signal pathway; Oxidative stress; Preeclampsia.

Fig. 1

Klotho was correlated with the development of PE in clinic and in vitro. A The contents of Klotho in PE patients’ serum were determined by ELISA analysis. The mRNA and protein levels of Klotho in PE patients’ placental tissues were respectively determined by performing B Real-Time qPCR and C Western Blot analysis. The human trophoblasts were subjected to hypoxia treatment, and D, E Real-Time qPCR and F, G Western Blot was used to detect the expression levels of Klotho protein in those cells in vitro. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times

Fig. 2

Overexpression of Klotho reversed the regulating effects of hypoxia on cell viability, apoptosis, invasion and senescence. A, B The human trophoblasts were subjected to hypoxia, and cell viability was determined by performing MTT assay. C The Annexin V-FITC/PI double staining method was employed to detect cell apoptosis of human trophoblasts. D Cell invasion abilities of HTR-8/SVneo and TEV cells were determined by performing Transwell assay. E Western Blot analysis was used to analyze the expression status of the cellular senescence-related proteins (p16 and p21) in human trophoblasts. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times

Fig. 3

Klotho regulated oxidative stress in PE models. A The MDA levels and B GSH/GSSG ratio in PE patients’ placental tissues were determined by their corresponding detection kits. C The expression levels of anti-oxidant proteins (Nrf2, SOD2 and NQO1) in PE patients’ placental tissues were detected by performing Western Blot analysis. D The MDA levels and E GSH/GSSG ratio in hypoxic human trophoblasts were respectively determined by their kits. F, G The human trophoblasts were subjected to hypoxia, and Western Blot analysis was performed to examine the expression status of nuclear Nrf2, total Nrf2, SOD2 and NQO1 in the cells. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times

Fig. 4

Silencing of Nrf2 re-triggered oxidative damages in Klotho-overexpressed hypoxic human trophoblasts. A, B The regulating effects of Nrf2 knockdown on the expression levels of nuclear Nrf2, total Nrf2, SOD2 and NQO1 were determined by performing Western Blot analysis. C The MDA levels and D GSH/GSSG ratio in human trophoblasts were determined by using their corresponding kits. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times

Fig. 5

Klotho regulated cell viability, apoptosis, invasion and senescence in hypoxic human trophoblasts by regulating the Nrf2/ARE signal pathway. A, B The cell viability of human trophoblasts was determined by using the MTT assay. C Cell apoptosis in human trophoblasts was determined by using the apoptosis detection kit. D Transwell assay was performed to examine cell invasion abilities of human trophoblasts. E The cellular senescence-associated proteins (p16 and p21) were examined by performing Western Blot analysis. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times

Fig. 6

Klotho regulated oxidative damages and cell mitosis in PE rat models in vivo. A ELSIA was used to examine Klotho concentrations in PE rats’ serum. The mRNA and protein levels of Klotho in PE rats’ placental tissues were respectively determined by performing B Real-Time qPCR and C Western Blot analysis. D The MDA levels and E GSH/GSSG ratio in PE rats’ placental tissues were determined by their commercial kits. F The expression levels of total Nrf2, SOD2 and NQO1 in PE rats’ placental tissues were determined by conducting Western Blot analysis. Real-Time qPCR was employed to examined the mRNA levels of G CDK2, H CDK6 and I Cyclin D1 in rats’ placental tissues. Each experiment was repeated at least for three times, and *P < 0.05 was considered as statistical significance. Each experiment was repeated at least for three times