| Objective:To explore the role of hydroxycitric acid(HCA)in renal stone injury and determine that HCA inhibits the formation of calcium oxalate renal stone induced by glyoxylic acid in rats.To reveal the influence mechanism of hydroxycitric acid on the occurrence of renal calculi,and to clarify that HCA mainly inhibits the formation of calcium oxalate calculi by enhancing tissue antioxidant capacity.It is revealed that hydroxycitric acid regulates calcium oxalate kidney stones by up regulating Nrf2 signal and enhancing tissue antioxidant capacity.To provide theoretical support for the research on the molecular mechanism related to the development of calcium oxalate renal calculi and provide a new idea for the prevension treatment of renal calculi.Methods:1.Collect the blood and urine samples of patients with renal calculi in urology department of the hospital,detect the biochemical indexes,and analyze the correlation.2.The rat model of calcium oxalate nephrolithiasis was established by intraperitoneal injection of glyoxylic acid.Then the rats were divided into control group,model group,hydroxycitric acid(HCA)group and citric acid(CA)group.The contents of BUN,Cr,UA and Ca2+were detected;Detect the content of OPN and SOD;HE staining was used to observe the crystallization and dilatation of renal tubular lumen;The mitochondria of glomerular podocytes,basement membrane and renal tubular epithelial cells in renal cortex were observed by transmission electron microscope.3.The rats were divided into control group,model group,hydroxycitric acid(HCA)group and HCA+Nrf2 knocked down(HCA+siNrf)group.The 24-hour urine volume,urinary ox,Ca2+ and OPN concentrations of rats in each group were detected,and the contents of serum BUN,Cr,UA and Ca2+were detected at the same time.The renal tissues of rats in each group were taken to detect the expression level of Nrf2,the activity of SOD and cat and the content of OPN;At the same time,the renal tissues of rats in each group were stained with he;Ki-67 and TUNEL staining were used to detect the proliferation activity and apoptosis of renal cells4.Human renal tubular epithelial cells(hK2)and rat renal tubular ductal epithelial cells(NRK-52E)were treated with oxalate to establish the cell model of calcium oxalate injury.The cell proliferation activity was detected by Ki-67 staining and the apoptosis level was detected by TUNEL staining;QRT PCR was used to detect the expression of oxidative stress related genes;The concentration of Ca2+cells was measured and the expression levels of Nrf2,SOD1,SOD2,SOD3 and cat were analyzed by Western blot.Construct effective siRNA,inhibit the expression of Nrf2(siNrf2)in HK2 and NRK-52E cells by RNAi technology,then treat the cells with sodium oxalate,the changes of the above experimental results were observed after knockout of Nrf2 and treatment of two kinds of cells with HCA and CA.Results:1.Urine volume was significantly correlated with sodium,creatinine and C-reactive protein,and positively correlated with oxalic acid and calcitonin.The content of oxalic acid in urine has a very significant positive correlation with urinary citric acid and oxalic acid,and has a significant positive correlation with potassium in blood.There was a significant positive correlation between urine creatinine content and blood potassium content,and there was a positive correlation between blood uric acid and blood sodium content.There was a positive correlation between blood calcium and blood sodium content,and a very significant positive correlation with CT value of imaging indexes.There was a significant negative correlation between blood sodium ion and potassium ion,a positive correlation with CT value,and a very significant positive correlation between C-reactive protein and calcitonin.2.The rat model showed that compared with the control group,the contents of BUN、Cr and UA in the model group were higher,the contents of OPN and SOD were lower,and the content of Ca2+had no significant difference.In HCA and CA groups,BUN,Cr and UA were significantly lower than those in model group,and the contents of OPN and SOD were higher.HE staining was used to observe the formation of calcium oxalate crystals in renal tubules,suggesting that HCA and CA can inhibit the formation of calcium oxalate crystals in rats,and the effect of HCA is stronger than that of CA.The observation of rat organelles by TEM suggests that the renal tubular epithelial cells and mitochondria in the model group are seriously damaged,and HCA can improve this kind of damage.3.After knocking down Nrf2 and adding HCA,the urine volume of rats decreased;There was no significant change in urine Ca2+concentration and serum Ca2+concentration before and after Nrf2 knockout;Compared with HCA group without knockdown of Nrf2,the contents of urinary ox,serum Cr,BUN and UA increased,while the concentration of urinary OPN decreased.The results of Ki-67 and TUNEL staining showed that after knockdown of Nrf2,HCA had no obvious effect on the decrease of renal cell proliferation activity and the increase of apoptosis caused by naox.4.The cell model showed that hydroxycitric acid had an effect on both HK2 and NRK-52E cell line models.The results of Ki-67 staining showed that the cell proliferation of the model group of HK2 and NRK-52E cell lines was low.The cell proliferation of the two cell lines treated with hydroxycitric acid was enhanced.The results of TUNEL staining showed that the apoptosis of the model group was greater than that of the treatment group(P<0.01).Both hydroxycitric acid and citric acid could reduce the apoptosis of the two groups,The effect of hydroxycitric acid on stone model was greater than that of citric acid.The results of HK2 and NRK-52E cell model treatment showed that the expression of oxidative stress related genes in calcium oxalate model group was higher than that in control culture group,but lower than that in hydroxycitric acid and citric acid treatment group,while the expression of oxidative stress related genes in citric acid treatment group was lower than that in hydroxycitric acid treatment group(P<0.01).Western blot showed that the expression of oxidative stress related proteins in stone model group was higher than that in normal control,lower than that in hydroxycitrate treatment and citrate treatment.At the same time,the expression of oxidative stress proteins in citrate treatment group was lower than that in hydroxycitrate treatment group,the difference was statistically significant(P<0.01).By knocking down Nrf2 in HK2 and NRK-52E cell treatment group and model group,the results showed that after knocking out Nrf2 signal,the effect of hydroxycitric acid treatment and citric acid treatment group on calcium oxalate kidney stones decreased significantly.In the calcium oxalate kidney stone model group with Nrf2 signal knockdown,even if hydroxycitric acid and citric acid were added,the level of related oxidative stress and cell level did not increase significantly.In the case of knockout of Nrf2,there was no significant difference in the expression of oxidative stress genes and proteins between the treatment group and the calcium oxalate kidney stone model group.Conclusions:1.Citric acid(CA)and hydroxycitric acid(HCA)inhibit the formation of calcium oxalate kidney stones in rats.The therapeutic effect of HCA is better than that of CA.2.HCA has a protective effect on sodium oxalate mediated injury of renal tubular epithelial cells;HCA up regulates the expression of oxidative stress related genes and proteins in sodium oxalate injury model of renal tubular epithelial cells.3.HCA up regulates the antioxidant level through Nrf2 antioxidant signal,enhances the expression of OPN,promotes oxalate metabolism,inhibits the formation of calcium oxalate kidney stones and calcium oxalate crystal induced renal function injury in rats. |
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