| Objective:Hydroxyapatite(HAP)is one of the important inorganic components that make up human bones and teeth.It has good biological activity and biocompatibility,and is a commonly used material for bone defect repair.However,the existence of HAP is not always beneficial,and most kidney stones are related to HAP.However,how HAP promotes the formation of kidney stones is currently rare.This study explored the effect of Nano-HAP on the apoptosis of renal tubular epithelial cells(HK-2 cells)and its mechanism,so as to understand the role and mechanism of Nano-HAP in the formation of kidney stones.Methods:1.We synthesized Nano-HAP using hydrothermal method according to the literature,and tested it with transmission electron microscope(TEM),scanning electron microscope(SEM),X-ray diffractometer(XRD)and solid-state fluorescence measuring instrument(PL)The materials were characterized.2.We selected human-derived renal tubular epithelial cells(HK-2 cells),and used tetramethylazozolium blue(MTT)colorimetry,flow cytometry,live cell / dead cell activity staining method,and intracellular Localization and other methods were used to study the effects of Nano-HAP on the survival rate,reactive oxygen species,apoptosis and apoptosis mechanism of HK-2 cells in vitro.Results:1.The morphology and size of Nano-HAP were measured by TEM and SEM.The results show that Nano-HAP is a rod with good dispersion,with a length of about 60 to100 nm and a height of about 12 nm.Analysis of the composition and structure of XRD showed that the synthesized material was pure HAP with good dispersibility.Nano-HAP blue light was measured by a solid-state fluorescence meter.Nano-HAP at different concentrations(0,5,10,20,40,80 μg / m L)were incubated with HK-2 cells for 24 h,48 h,and 72 h.The MTT results showed that Nano-HAP was dose-and time-dependent.Sexually inhibits the activity of HK-2 cells.Nano-HAP at different concentrations(0,5,10,20,40,80 μg / m L)was incubated with HK-2 cells for 24 h.The results of live / dead cell staining also suggested that Nano-HAP could inhibit cell proliferation.When Nano-HAP was at 80ug/ml,HK-2 cells showed a large amount of shedding and apoptosis,and the results of MTT and live/dead cell staining were basically the same.3.Nano-HAP at different concentrations(0,10,20,40μg/m L)were incubated with HK-2 cells for 6h,24 h,6h apoptosis results.Apoptosis results had no significant effect on apoptosis.24 h apoptosis results showed that Nano-HAP can induce apoptosis of HK-2 cells,which increases with increasing concentration and is dose-dependent.Nano-HAP(40 μg/m L)was incubated with HK-2 cells for 6 h.It was observed by confocal microscopy that Nano-HAP was mainly localized in HK-2 cells lysosomes.Nano-HAP was incubated with HK-2 cells for 6 h at different concentrations(0,10,20,40 μg/m L).Nano-HAP induced increased ROS levels,increased lysosomal membrane permeability,and mitochondrial membrane in HK-2 cells.The potential decreases and becomes concentration-dependent.Nano-HAP at different concentrations(0,10,20,40 μg/m L)was incubated with HK-2 cells for 6 hours.Caspase-3 levels increased first and then decreased.When Nano-HAP was at 40 μg/m L,The decrease in activity may be related to cell necrosis at higher concentrations.Conclusions:Nano-HAP induces apoptosis through lysosomal and mitochondrial pathways.Our research will provide some valuable data for the future role of Nano-HAP in the formation of kidney stones.It is worth noting that Nano-HAP did not cause apoptosis when it was applied to HK-2 cells for 6 hours,but a large amount of reactive oxygen species was generated,mitochondrial membrane potential decreased,lysosomal membrane permeability increased,and caspase-3 activity appeared A lot of changes.Apoptosis and the factors that cause apoptosis do not appear at the same time. |
