| ObjectiveThis study aims to establish the rat model of calcium oxalate kidney stone and culture of renal tubule epithelial cells in vitro.Through different concentration of the drug intervention,the changes of urinary biochemical,renal tissue calcium oxalate crystallization and immunohistochemistry were observed.Therefore,the mechanism and target of the treatment of renal stones by the Paishi granule are further discussed,so as to provide theoretical basis for the prevention and treatment of urinary stones.Method Experiment one: Preparation of rat model of calcium oxalate kidney stone.40 rats were fed,drinking water,and adapted to the environment for a week.According to the random sampling method,it is divided into: normal control group(group A),0.75 % ethylene glycol + 0.75 % ammonium chloride solution lavage group(group B),1 % ethylene glycol+1% ammonium chloride solution lavage group(group C),1% ethylene glycol +2 % ammonium chloride solution lavage group(D)In the group,there were 10 rats in each group.The experimental period is 28 days(4 weeks).To observe the excretion of uric acid,urinary calcium,urinary NAG enzyme,urine ?2 microglobulin,renal function test,and to observe the deposition of crystals in renal tissue.Experiment two:60 rats were fed adaptively for one week,and were divided into normal control group,model group,Paishi Granule low dose group,Paishi Granule Middle dose group,Paishi Granule high dose group,potassium citrate treatment group,each with 10 rats.The model-making and model-replication methods used 1% ethylene glycol free drinking water and 2% ammonium chloride solution as the stone-forming agent for calcium oxalate calculus.In addition to the normal group,the other groups were given lithotripsy for an experimental period of 4 weeks(28 days).To observe the general condition of rats and compare the indexes of urine biochemistry,renal function and blood biochemistry among different groups.Experiment three:Extraction of the kidney of each group of rats,pathological section,HE staining,Vonkossa calcium nodule staining,kidney pathological changes,quantitative score comparison calcium crystals.Experiment four:The expression of TGF-?1,p38 MAPK in the renal tissue of rats were observed by immunohistochemistry,and the levels of TGF-?1,47 phox and P38 MAPK were detected by RT-PCR.Experiment five:The expression of Claudin-14 and p38 MAPK in the renal tissue of rats was observed by immunohistochemistry and the expression of Claudin-14 and p38 MAPK in the renal tissue of rats was detected by Western blotting.Experiment six:50 rats,adaptive feeding after a week,according to the random method divided into: normal group,model group,Paishi Granule low dose group,Paishi Granule Middle dose group,Paishi Granule high dose group,each group has 10 rats.The dosage of Chinese herbal medicine was 1ml/100 g,the concentration of the drug was: high concentration 3.6g/ml,medium concentration 2.4 g/ml,low concentration metric 1.2 g/ml,continuous infusion of 3D,1 H after the last infusion,blood from the abdominal aorta,separation of serum,and storage in-80 ? refrigerator.50 rats,after a week of adaptive feeding,they were randomly divided into normal group,model group,Paishi Granule low dose group,Paishi Granule Middle dose group,Paishi Granule high dose group.The treatment components were replaced with 5ml DMEM solution containing 5 mg/LCOM and 5 doses of serum containing 5 mg/L and 3 groups of Paishi Granule.After 48 h,the culture was removed,the cells were taken and the total protein and RNA were extracted.Result(1)The results of the experiment showed that compared with the normal group,the excretion of uric acid and calcium in the three concentrations of ethylene glycol,ammonium chloride solution irrigation group increased,and only 1 % of ethylene glycol and 2 % of ammonium chloride in the gastric irrigation group.The increase has statistical significance(P 0.01).Three concentrations of ethylene glycol,ammonium chloride solution in the gastric group of urine NAG enzyme,urine ?2-MG compared with the normal group,of which 1 % ethylene glycol,1 % ammonium chloride group and 1 % ethylene glycol,2 % ammonium chloride Group has statistical significance(P 0.01).0.75 % ethylene glycol + 0.75 % ammonium chloride solution lavage group,1 % ethylene glycol + 1 % ammonium chloride lavage group two groups of glomerulus,renal tubules are almost normal,no deposits of calcium salt crystals were seen in renal tissue.Ca Ox crystals were observed in the kidney of rats with 1 % ethylene glycol+2% ammonium chloride solution in the stomach irrigation group.(2)The results showed that urinary oxalic acid and urinary calcium excretion were significantly higher in the model group than in the normal group(P<0.01).Compared with the model group,the significant decrease of urinary oxalic acid and urinary calcium was observed in the high dose group and potassium citrate group(P<0.01).Comparison of Scrs: There was a significant increase in the model group,the three dosage groups of Paishi granule and the group of potassium citrate(P<0.01).Compared with the model group,Scrs in the high dose group and potassium citrate group were significantly decreased and had significant difference(P <0.05).(3)The results of experiment 2 showed that the kidney of the model group was dull and lustrous,swollen,and the cut surface was pale,and there were granulocyte like substances scattered in the distribution of protuberant,some of which expanded and the renal parenchyma became thin.Compared with the normal group,the crystal score of the model group and the lower dose group of the pellet granule increased significantly and was statistically significant(P < 0.01).Compared with the model group,the scores of the high-dose group and the potassium citrate group were significantly reduced(P<0.01).There was also a significant decrease in the dose group and the model group(P<0.05).(4)The results showed that TGF-?1 and p38 MAPK proteins were expressed in the renal tissue of normal rats,and the color of immunostaining was weak.In the model group,TGF-?1,p38 MAPK were expressed in renal tubules at high levels,and the intensity and distribution of immunohistochemical staining were significantly enhanced.The expression of TGF-? 1 and p38 MAPK protein in glomerular and interstitial tissues of rats in high dose group decreased significantly(P < 0.01).(5)The results of experiment 4 showed that in normal group of rats,the expression of Claudin-14 protein was small and the color of immunostaining was weak.In the model group,Claudin-14 protein was expressed at high level in renal tubules of rats,and the intensity and distribution of immunohistochemical staining were significantly enhanced.The expression of Claudin-14 protein in glomerular and interstitial tissues of rats in high dose group decreased significantly(P 0.01).The expression of p38 MAPK protein in the renal tissue of rats in the model group was significantly increased(P < 0.01).p38 MAPK decreased significantly in the small dose group than the model group,decreased significantly in the middle and high dose group(P<0.05).(6)The results of experiment 5 showed that the expression of Claudin-14 and p38 MAPKm RNA in NRK52 E cells in model group was significantly enhanced(P < 0.01).The expression of low-dose,medium-dose Claudin-14,and p38 MAPKm RNA was higher than normal group,but the difference was significant.The high dose group expression of Claudin-14 and P38 MAPKm RNA was significantly higher to the normal group and decreased significantly compared with the model group(P< 0.05).Conclusion(1)Calcium oxalate stone model method: 1 % ethylene glycol free drinking water and 2% ammonium chloride solution gastric injection molded successfully,safe and effective.(2)The drainage agent can reduce the excretion of urinary oxalic acid and urinary calcium in rats with calcium oxalate stones,reduce the blood creatinine,and protect renal function.(3)The excretion of urine oxalic acid and urine calcium in rats with calcium oxalate calculus can be reduced,blood creatinine can be reduced and kidney function can be protected.Calcium oxalate stone in the renal tissue of rats with a large number of calcium crystal deposition,platoon stone granule can reduce calcium granule,promote stone expulsion.(4)The key subunits of TGF-?1 and NADPH oxidase increased significantly in renal tissue of rats with calcium oxalate calculus.The reaction of renal tubule epithelial cells to oxidative stress of calcium oxalate is related to inflammation.(5)The expression of Claudin-14 in rats with calcium oxalate calculus was significantly enhanced by RT-PCR and WB,and the excretion of urinary calcium was positively correlated with that of Claudin-14.There was a significant decrease of Claudin-14 in the high dose group,which may be related to the expression reduction of Claudin-14,and the urinary calcium excretion reduction.(6)The expression of p38 MAPK was obviously stronger than that of the normal group,which confirmed the activation of p38 MAPK signal pathway.The mechanism may be oxidative stress,reactive oxygen species(ROS),TGF-Beta 1 as the upstream stimulation of P38 MAPK signal pathway activation,P38 MAPK signal pathway can stimulate the production of reactive oxygen species,interleukin-6 and other inflammatory factors.The expression of p38 MAPK decreased in the high dose group,which may be associated with the inhibition of p38 MAPK signal.It provides the theoretical basis for treating kidney stones better with the powder. |
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