Abstract:

Objective To discuss the expression of Klotho in placenta exosomes （Exo）of the patients with pre-eclampsia （PE），and to clarify its effect on the oxidative stress in the vascular endothelial cells. Methods The clinical data of 40 pregnant women including 20 with normal pregnancy（NP） women （NP group） and 20 PE patients （PE group） were collected. The placenta Exo in peripheral blood of the patients in two groups were isolated， and the oe-Klotho and oe-NC plasmids were transfected into the human chorionic trophoblast cells（HTR-8/SVneo）， respectively， and were regarded as oe-Klotho group and oe-NC group.The expression levels of Klotho mRNA and protein in placenta Exo and the HTR8/SVneo cells in two groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method and Western blotting method. The human umbilical vein endothelial cells （HUVECs） with good growth status were taken and divided into PE-Exo group （co-cultured with placenta Exo from the patients with PE），NP-Exo group （co-cultured with placenta Exo from the NP subjects）， oe-Klotho-Exo group （co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-Klotho）， and oe-NC-Exo group （co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-NC） according to the sources of Exo.The expression levels of exosomal marker proteins CD63， TSG101， and placenta Exo-specific marker PLAP protein were identified by transmission electron microscope （TEM） and Western blotting method； the levels of nitric oxide （NO）， reactive oxygen species （ROS）， and malondialdehyde （MDA） and the activities of superoxide dismutase （SOD） in the HUVECs in various groups were detected by enzyme-linked immunosorbent assay （ELISA） method；the expression levels of endothelial nitric oxide synthase （eNOS） mRNA in the HUVECs in various groups was detected by RT-qPCR method；the expression levels of eNOS protein in the HUVECs in various groups were detected by Western blotting method. Results Compared with NP group， the systolic and diastolic blood pressures of the patients in PE group were increased （P<0.05）， the body weight of newborn and placenta weight were significantly decreased （P<0.05 or P<0.01）. The TEM observation results showed that the placenta Exo derived from the subjects in both NP and PE groups being round or oval vesicular discoid structures with complete membrane and similar shape， with a diameter of 50—100 nm. The Exo from the subjects in both groups highly expressed Exo marker proteins CD63 and TSG101， and placental Exo-specific protein PLAP. The nanoparticle tracking analysis （NTA） results showed that the particle size of Exo was 50—200 nm， suggesting that the vesicles isolated from the peripheral blood were derived from placental Exo. The TEM， Western blotting， and NTA results confirmed that the vesicles from oe-NC group and oe-klotho group had typical Exo structures， molecular markers， and particle size characteristics. The Exo with green fluorescence expression could be observed in the HUVECs， suggesting that the placenta Exo could be taken up by the HUVECs. The ELISA results showed that compared with NP-Exo group， the level of NO and activity of SOD in PE-Exo group were significantly decreasd（P<0.05 or P<0.01）， and the levels of ROS and MDA were significantly increased（P<0.05 or P<0.01）； compared with oe-NC-Exo group， the level of NO and activity of SOD in the HUVECs in oe-Klotho-Exo group were increased （P<0.05）， and the levels of ROS and MDA were significantly decreased （P<0.05 or P<0.01）. The RT-qPCR results showed that compared with NP group， the expression level of Klotho mRNA in placenta Exo of the patients in PE group was significantly decreased （P<0.01），and the expression level of eNOS mRNA in the HUVECs was significantly decreased （P<0.01）； compared with oe-NC group， the expression level of Klotho mRNA in Exo in the trophoblast cells in oe-Klotho group was significantly increased （P<0.01）； compared with oe-NC-Exo group， the expression level of eNOS mRNA in the HUVECs in oe-Klotho-Exo group was significantly increased （P<0.01）. The Western blotting results showed that compared with NP group， the expression level of Klotho protein in placenta Exo of the patients in PE group was significantly decreased （P<0.01）； compared with NP-Exo group， the expression level of eNOS protein in the HUVECs in PE-Exo group was significantly decreased （P<0.01）； compared with oe-NC group， the expression level of Klotho protein in Exo in the trophoblast cells in oe-Klotho group was significantly increased （P<0.01）； compared with oe-NC-Exo group， the expression level of eNOS protein in the HUVECs in oe-Klotho-Exo group was significantly increased （P<0.01）. Conclusion The placenta Exo from the PE patients may inhibit the production of NO in the HUVECs and promote the oxidative stress to impair the endothelial cell function. The over-expression of Klotho in Exo in the trophoblast cells can reduce the production of NO and level of oxidative stress in the HUVECs.

Key words: Klotho protein, Pre-eclampsia, Exosomes, Vascular endothelial cell, Oxidative stress