In Vitro Simulation Of Effects Of Cell Injury And Polysaccharides Intervention On Vascular Calcification And Kidney Stone Formation

Kidney stones and vascular calcification are common diseases in the human body.The etiology of both is due to ectopic calcification.Kidney stones are caused by the formation and deposition of crystals on the surface of renal epithelial cells in the kidney.Vascular calcification is due to the formation of crystals on the surface of vascular smooth muscle cells and the deposition of crystals on the surface of blood vessels,which causes the phenotypic transformation of cells in blood vessels,leading to the conversion of smooth muscle cells in blood vessels to osteoblasts.There are few studies on the effects of calcium deposition on cells before and after cell injury.Therefore,in chapter two,we studied the difference between the effects of hydroxyapatite on cell calcification before and after high phosphorus damage to A7R5 cells.For kidney stones,the drug treatment is still in the research stage.Polysaccharides are natural oxidants,contain a large number of anions,and their structures are similar to those of the urine stone inhibitor GAG.Therefore,Therefore,chapters 3 and 4 study the inhibition of polysaccharides on crystals and the protection of renal epithelial cells(HK-2)to provide new enlightenment for the search for drugs against kidney stone.The first chapter briefly summarizes the formation of vascular calcification,the effects of high phosphorus and hydroxyapatite on vascular calcification,the formation mechanism of kidney stones and the mechanism of polysaccharides inhibiting kidney stones.In chapter 2,vascular calcification(VC)is the main cause of death in patients with chronic kidney disease(CKD).The main product of VC is hydroxyapatite(HAP).This study simulated the high phosphorus(Pi)environment in CKD patients.Nano-HAP crystals were used to damage the rat aortic smooth muscle cells(A7R5)which have been pre-damaged with different concentrations of Pi solution,to compare the difference of HAP-induced calcification in A7R5 cells before and after injury by high Pi.After A7R5 cells were damaged by high-Pi,HAP caused cell viability and lysosomal integrity declined,lactate dehydrogenase(LDH)release and reactive oxygen species(ROS)increased,caused more serious cell damage,and the ability to internalize HAP crystals declined,the cells ability to adhere to HAP increased,the amounts of calcium deposition on the cell surface,alkaline phosphatase(ALP)activities increased and the expression of osteopontin(OPN)and Runt-related transcription factor 2(Runx2)increased,HAP-induced osteogenic transformation was enhanced.In chapter 3,the effects of four green tea polysaccharides(TPSs)with molecular weight of 10.88(TPS0),8.16(TPS1),4.82(TPS2)and 2.3 k Da(TPS3),respectively,on the crystallization of calcium oxalate(Ca Ox)crystal and protective effect on human proximal tubular epithelial cells(HK-2)were comparatively studied.X-ray diffraction,Fourier transform infrared spectroscopy,and scanning electron microscopy results revealed that TPS1,TPS2 and TPS3 can increase the percentage of dihydrate crystalline phase in Ca Ox crystals and reduced the size of Ca Ox monohydrate crystals.TPSs increased the absolute value of Zeta potential of Ca Ox crystal and inhibited crystal nucleation and aggregation.The nucleation inhibition rate and aggregation inhibition rate of TPS1,TPS2,TPS3 to Ca Ox crystallization were 56.67%,75.52%,52.92%,and 22.34%,47.59%,21.59%,respectively.TPSs protected cells can alleviate oxidative damage.Pre-protection of TPSs can alleviate oxidative damage of HK-2 cells caused by oxalate,increase cell viability,protect cell morphology,and reduce lactate dehydrogenase release and reactive oxygen levels.In chapter 4,the pre-protective effects of polysaccharides from Porphyra yezoensis(PYP1,PYP2,PYP3 and PYP4)with molecular weights of 576.2,105.4,22.47 and 3.89 k Da respectively on oxidative damage of human renal proximal epithelial cells(HK-2)and the difference of adhesion and endocytosis of HK-2 cells to calcium oxalate monohydrate(COM)crystals before and after protection were studied.The results showed that PYPs could effectively reduce the oxidative damage of oxalic acid to HK-2 cells.Under the pre-protection of PYPs,the cell vitality increasesed,the cell morphology was improved,the level of reactive oxygen species(ROS)decreased,the mitochondrial membrane potential increased,the S phase arrest of cells was inhibited,the cell apoptosis rate decreased,the turnover of phosphatidylserine(PS)decreased,the amount of crystals adhered to the cell surface decreased,but the amount of endocytotic crystal increasesd.