The Role And Mechanism Of Theaflavin In Inhibiting Calcium Oxalate Kidney Stone Formation Through Antioxidant Stress

Part I The effect of theaflavin on reducing calcium oxalate crystal deposition and renal tubular epithelial cell injury by regulating oxidative stressObjective:Crystal-induced cellular oxidative stress injury plays a key role in the early stage of calcium oxalate（Ca Ox）kidney stone formation,tubule epithelial cell injury could increase the expression of cell surface adhesive proteins,which further promoted the adhension and aggregation of Ca Ox crystals.This study aimed to explore the role and influence of theaflavin（TF）on crystal-cell injury and Ca Ox crystal deposition in kidney.Methods:The mice model of crystalline kidney injury was established by intraperitoneal injection of glyoxylic acid,30 male C57BL/6 mice（6-8 week old）were randomly divided into 5 groups:normal control group,crystallized renal injury model group,low-dose TF treatment group（20 mg/kg）,medium-dose TF treatment group（50 mg/kg）,and high-dose TF treatment group（100 mg/kg）.Polarized light microscope and Pizzolato staining were used to observe Ca Ox crystal deposition in renal tissues of mice.Tubular injury and cell apoptosis were assessed by PAS staining and TUNEL fluorescence in situ staining.ROS production level in renal tissues was evaluated using DHE method.IHC staining was used to detect the protein expression of NOX2,SOD2,CD44,and OPN in the mice kidneys.Serum creatinine and urea nitrogen of mice were measured using commercial kits.In cell experiments,the proliferation activity of HK-2 cells was detected by CCK-8 method to determine the concentration of TF treatment,and then the cells were divided into four groups based on treatment methods:normal control group,COM treatment group,20μg/ml TF treatment group,and 50μg/ml TF treatment group.Cell apoptosis,ROS production,H₂O₂,LDH,MDA,and SOD levels,stone-related protein CD44 and OPN expression,and crystal adhesion to tubular epithelial cells were detected and evaluated in each group.Results:We successfully established the mouse model of crystalline kidney injury.The amount of Ca Ox crystal deposition,the degree of tubular injury,the number of cell apoptosis,the level of ROS production,the expression levels of CD44 and OPN,and the levels of serum creatinine and urea nitrogen in the model group mice were significantly increased compared with those in the normal control group.However,in TF treatment groups,the levels of all above indexes were obviously lower than those in the model group,and the reduction effect was positively correlated with the treatment dose.In cell experiments,the concentration of TF≤50μg/ml had no significant effect on the activity of HK-2 cells.After COM crystal treatment,the number of cell apoptosis,ROS production,H₂O₂,LDH,MDA,and SOD levels,the expression of CD44 and OPN,and crystal adhesion amounts significantly increased compared to those of normal control group;but after TF pretreatment,the levels of these indexes were reversed.The effect of 50μg/ml TF was better than that of 20μg/ml TF.Conclusions:TF could inhibit oxidative stress and improve crystalline kidney injury,reducing the adhesion and deposition of Ca Ox crystals,and thus may contribute to the inhibition of kidney stone formation.Part II The mechanism of theaflavin alleviating oxidative injury of kidney induced by calcium oxalate crystals through mi R-128-3p/SIRT1 axisObjective: According to the results of Part I,we confirmed the effect of theaflavin（TF）in reducing calcium oxalate（Ca Ox）crystal deposition and alleviating tubular epithelial cell damage.In this part,we aimed to explore the possible mechanism of TF’s protective effect on Ca Ox crystal induced renal oxidative damage,and especially to discuss the role of mi R-128-3p/SIRT1 axis.Methods: Firstly,the expression of SIRT1 in the kidneys of mice was detected by IHC and q PCR,and the expression of mi R-128-3p was detected by FISH and q PCR.Then,the expression relationship between SIRT1 and mi R-128-3p was evaluated by Pearson correlation coefficient analysis.In cell experiments,the expression levels of SIRT1 and mi R-128-3p after COM crystals with or without TF pretreatment were detected respectively,and the regulatory effect of mi R-128-3p on SIRT1 was determined using dual luciferase reporting assay,q PCR,and western blot.Finally,mi R-128-3p mimic and Agomir-128 were used to increase the expression of mi R-128-3p in HK-2 cells and the kidneys of mice,respectively.A series of experiments in vivo and in vitro were conducted to observe the effects of TF on Ca Ox crystal induced oxidative stress,crystalline kidney injury,and crystal adhesion and deposition on renal tubular epithelial cells by regulating mi R-128-3p/SIRT1 axis.Results: Firstly,we observed decreased expression of SIRT1 and increased expression of mi R-128-3p in the kidneys of glyoxylic acid-induced crystal group mice compared to those in normal control group,while TF treatment could upregulate SIRT1 expression and downregulate mi R-128-3p expression.Pearson correlation coefficient analysis confirmed that the expression of SIRT1 was negatively correlated with mi R-128-3p expression.In cell experiments,we also found that COM crystal treatment could decrease the expression of SIRT1 and increase the expression of mi R-128-3p,while TF treatment could reverse their expression changes.Subsequently,dual luciferase report assay,q PCR,and western blot results indicated that mi R-128-3p could directly target the 3’-UTR of SIRT1 to inhibit its expression.Finally,further results in cell and animal experiments also confirmed that mi R-128-3p activation could aggravate Ca Ox crystal induced oxidative stress,cell apoptosis,and crystal adhesion,and also partially reverse the upregulation of SIRT1 expression and the protective effect on crystalline kidney oxidative injury by TF supplement.Conclusions: TF could increase the expression of SIRT1 but decrease the expression of mi R-128-3p,to inhibite renal oxidative stress and reduce the adhesion and deposition of Ca Ox crystal.The regulation of mi R-128-3p/SIRT1 axis might be an important pathway for TF to alleviate renal injury caused by Ca Ox crystal and exert renal protection function.TF is expected to provide a new idea and choice for the prevention and treatment of Ca Ox kidney stones.