| The mechanism of infant urinary calculus caused by the milk powder containing melamine and the component of the calculus are unclear.There are no specific detoxicating methods for the clinical treatment of these calculus,except for conventional therapies,such as drug treatment,surgical intervention and extracorporeal lithotripsy.It is difficult for the implement of treatment to heal the urinary calculus involved indication and complication, such as renal injury,because the patients are usually infants which intensify the difficulty. Moreover,radical treatment and prevention of recurrence are still tough problems for the conventional therapies.Chinese Medicine accumulates rich experience and abundant traditional prescriptions on the treatment of urinary calculus.Animal models of human disease provide experimental evidences for the improvement of disease treatment techniques and methods.So animal models of human disease have close relation with the development of clinic medicine and basic theory of biomedicine.Although new diseases and their pathogenic factors perplex the health of human,it promote the innovation and development of the research of human disease animal model.Therefore,it is very important to establish the animal model of urinary caused by melamine.1.ObjectiveThe purpose of this research was to explore the method of creating animal models for urinary calculus induced by melamine.This method will provide an innovative model for the basic research of preservation and treatment of urinary calculus.This research will assess the detoxification of San Jin Tang to the urinary caused by melamine in KM mice.2.Methods2.1 The research in acute toxicity of melamine.The chemically pure preparation of melamine whose purity is bigger or equal to 90%was used in this experiment.Dosage choice of melamine was based on the LDm and LDn from reference and preliminary test. KM mice(18～22 g;half male and half female) were divided by 0.8 class width into several groups.Implement of acute toxicity test was achieved by administration of different dosage to each group respectively by gavage.The purpose of this experiment was to assess the hazard of melamine and provide theory foundation for toxic mechanism. 2.2 The study on SD rat urinary calculus induced by high dosage of melamine.Rats were administered various dosages of melamine by gavage.Crystallization,calculus formation and pathological changes in kidney,metanephric duct and bladder,were observed at various stages.Biochemical indicator of blood were also inspected.190 SPF SD rats(60 days old,half male and half female,213～227g) were divided according to melamine dosage to the rats with dosage of 0.5,1.0,1.5,2.0,2.5 g·kg-1 and control group(34 rats per drug group and 20 rats per control group).Rats in control group were administered 10 g·L-1 methylcellulose distilled water by gavage.Morphology changes of kidney,metanephric duct and urinary bladder were observed by visual inspection and stereomicroscope.Weight and viscera index of kidney and urinary bladder in each group were compared.The changes of contents for CREA,BUM,UA,Ca,P,Mg were detected.2.3 The study on SD rat urinary calculus induced by low dosage of melamine.130 SPF SD rats(at 60 days old,200±24g,half male and half female) were divided into 5 drug groups(n =20),1 blank control group(n=20 ) and 1 solvent control group(n=10).Dosages of melamine administered to the drug groups by gavage were 0.05,0.1,0.2,0.3,0.4 g·kg-1. The blank control group rats were administered aseptic water by gavage 2mL.d-1.The solvent control group rats were administered 10 g·L-1 methylcellulose distilled water 2mL·d-1 by gavage.Morphology changes of kidney,metanephric duct and urinary bladder were observed by visual inspection and stereomicroscope.Weight and viscera index of kidney and urinary bladder in each group were compared.The changes of contents of CREA,BUM,UA,Ca,P,and Mg were detected.2.4 The observation of effect of melamine on SD rats kidney.Weight and viscera.index of heart,liver,spleen,lung,kidney,urinary bladder from animals in experiment 2 and 3 were recorded after 20 and 30 administration days respectively.The histopathological changes induced by melamine were also observed.2.5 The study on detoxification of San Jin Tang to the urinary caused by melamine in experimental animals.70 SPF KM mice(18～22 g,half male and half female) were divided into high dosage of San Jin Tang administered preventive group,middle dosage of San Jin Tang administered preventive group,low dosage of San Jin Tang administered preventive group,San Jin Tang treatment group,melamine model group,blank control group and solvent control group(n=10,half male and half female).Dosages of San Jin Tang administered to preventive groups were 28.4,14.2 and 7.08g·kg-1.The preventive groups were administered San Jin Tang for 2 days after administration of 1.0g.kg-1 melamine 2 days.The treatment group rats were administered San Jin Tang(14.2g·kg-1) for 2 days after administration of 1.0g·kg-1 melamine 2 days.Solvent control group rats were administered 10 g·kg-1 methyl cellulose distilled water by gavage 40mL·kg-1.The blank control group rats were administered aseptic water(40mL·kg-1) by gavage.General symptom of the mice was observed during the experiment.The contents of melamine were detected in kidneys and lungs from each group.This research used the National Standard GB/T22388-2008,to detect the contents of melamine in kidneys and lung.3.results3.1 The LD50 of melamine to KM mice by gavage was 9366 mg·kg-1.and the confidence limit of LD(50) was 8149～10761 mg·kg-1.According to the pathological observation,there were no crystallization,but inflammatory cells infiltration in kidney,liver and lung.And there were damaged micrangium,congestions and stagnant blood in lung tissue.3.2 The informations of high dosage melamine inducing SD rats urinary calculus:The ratio of rats successively administrated 10 days inducing calculus of the bladder was 9% (4/47) and the ratio of rats successively administrated 20 days inducing calculus of the bladder was 37%(11/30) and inducing ureteral calculus was 93%(28/30).The kidneys of each group rats successively administrated 10 and 20 days were swelling and isabel. And there were trab-like yellow precipitates in all the kidneys,and some kidneys had punctiform or lamellar hemorrhagic focus.The yellow precipitates in the kidneys of each group rats successively administrated 20 days began to become lamellar or granular,and there were yellow comma aureole between cortex and medulla.The color of the kidneys cross section of rats successively administrated 30 days and 20 days was nearly the same, but the yellow precipitates of the former collected to the renal cortex and the trab-like yellow precipitates reduced in renal medulla.One rat administrated 30 days and without drug 24 days in each group with dosage of 0.5 and 1.5g·kg-1 had changes after anatomy examination.And the changes of the kidneys cross section of rats were similar to that of 30d rats.But two rats had no calculus in their ureters and bladders.The kidneys weight and viscera index of the rats administrated 10,20 and 30 days were all 2 to 3 times higher than the control group.The concentration of CREA,BUN,P and Mg in blood significantly increased and the concentration of Ca slightly increased.3.3 The informations of the urinary calculus induced by low dosage of melamine to rats: The concentration of UN in rats administrated 20 days with dosage of 0.5～1.5g·kg-1 was a little higher than that of the blank control groups.But in the groups administrated 30 days,the concentration of Cr and UC was a little higher than that of the blank control groups.The concentration of Ca,P and Mg in blood to every group administrated 20 days with dosage of 0.5～1.5g·kg-1 was low.But in the group administrated 30 days with dose of 0.05,0.2,0.3,0.4 g·kg-1,the left kidney had higher weight than that of the blank control groups.The size and color of kidney were very near to that of the blank control groups and there were no calculus and yellow precipitates.But on the juncture between medulla and cortex in some kidneys of rats,there were punctiforrn or lamellar hemorrhagic focus.There were no calculus on the ureter.Part of mucous membrane of urinary bladder of rats had hyperemia and part of male rats had vesical calculus.The ratio for male rats administrated 20 days was 52%(13/25) and 30 days was 56%(14/25),The ratio of vesical calculus for rats with dose of 0.3 g·kg-1 administrated melamine 30 days was high to 70%.The vesical calculus were yellow-white and white.3.4 The effect of melamine to SD rat3.4.1 The observation of the organ weight and viscera index of rats administrated melamine 20 days:The index of kidney,liver and spleen of rats with dosage of 0.5～1.5g·kg-1 had significant difference compared with the group of dosage 0.05～0.4g·kg-1,blank control group and solvant control group.But the dosage of 0.05～0.4g·kg-1 had no significant difference compared with blank control group and solvant control group.The left lung index of rat with dose of 1.0g·kg-1 had significant difference compared with the groups with dose of 0.05-0.4g·kg-1,blank control group and solvant control group.The groups with dosage of 0.5 and 1.5g·kg-1 also had significant difference compared with the groups with dose of 0.2g·kg-1 and solvant control group.The weight of heart of rats with dose of 0.1g·kg-1 were higher than that of the groups with dosage of 0.5,1.0 and 1.5g·kg-1 and had significant difference compared with the groups solvant control group(p＜0.01).There were no changes in the weight and index of the bladder.3.4.2 The observation of the organ weight and viscera index of rats administrated melamine 30 days:The weight and index of kidneys and bladder of rats for the group with dosage of 0.5～1.5g·kg-1 had significant difference compared with the groups with dosage of 0.05～0.4 g·kg-1,blank control group and dissolvant control group.But the group with dose of 0.05～0.4 g·kg-1 had no significant difference compared with blank control group and solvant control group.The right lung of rats with dosage of 0.4g·kg-1 had significant difference(p＜0.01) compared with the blank control group.The weight of liver of rats with dose of 0.2 g·kg-1 had statistical significance compared with the blank control group (p＜0.01).The weight of spleen of every administration group excepting the group with dose of 0.5g.kg-1 and 1.0 g·kg-1 increased slightly and there were no statistical significance between every administration group and the blank control group.There were statistical significance between the administration group with dosage of 0.1,0.3,0.4g·kg-1 and the blank control group on the heart index.3.4.3 histopathological observation:HE stainig pathological result showed that the organization structure of kidneys,liver,spleen,heart and bladder were normal in blank and solvant control group,but there were different degrees of tissue damages and pathological changes in kidneys,liver,spleen of the drug groups.In the rats administrated 20 and 30 days with dosage of 0.5 to 1.5g·kg-1,the anatomy test showed that there were renal tubular necrosis or hydropic degeneration in renal cortex.And some renal tubular epithelial cells fell off.There were inflammatory cell infiltration and brown crystallization in stroma and renal tubular.Focus-like-gathering crystallization appeared in pelvis mucosa of some rats, and some mucous membrane was damaged.The kidneys of rats administrated 20 and 30 days with dosage of 0.05～0.4g·kg-1 had slight pathological changes and parts of coagulation necrosis or hydropic degeneration in renal cortex,but the renal medulla was normal.The liver cells of rats with dosage of 0.5～1.5g·kg-1 had hydropic degeneration and a little cells infiltration.The splenic cord and splenic sinusoid had many hemosiderin cells and the splenic sinusoid showed mid-ranged extension and congestion.The lung of rats had mild inflammation.The spleen,liver and lung of rats with dose of 0.05～0.4 g·kg-1 had very little inflammatory cell infiltrate and the histopathological observation of heart and bladder in drug group showed that there were no abnormal changes.3.5 The research for the effect of Chinese Medicine San Jin Tang on the clearance of melamine:There was residual melamine in the kidneys and lung of KM mice after administration for 24 hours.In general the content of melamine in kidney was higher than that in lung.The content of melamine in kidneys and lung for melamine animal models were the highest which was 327ng·g-1 and 177.5ng·g-1 respectively.The preventive use of high dosage San Jin Tang had obvious effects on cleaning up the melamine in kidneys and lung,and the test suggested that the content of melamine in kidneys and lung had reduced to 58ng·g-1 and 28.2ng·g-1.The effect of San Jin Tang on cleaning up the melamine in kidneys and lung from high to low was preventive use of high dosage San Jin Tang> preventive use of middle dosage San Jin Tang>preventive use of low dosage San Jin Tang > treating use of San Jin Tang.4.conclusion4.1 The LD50 of melamine to KM mice by gavage was 9366 mg·kg-1.When LD50>5000 mg·kg-1,it was micro-toxic or non-toxic according to the national grade standard of drug acute toxicity(melamine was administrated once for all).Inflammatory reaction of kidney, liver and lung was caused by melamine.4.2 After the SD rats were successively administrated 0.5～2.5g·kg-1 melamine 30 days, there were calculus in the bladder and ureter,and the yellow lamellar or granular precipitates appeared on the kidneys whose function was damaged.4.3 After the SD rats were successively administrated 0.05～0.4g·kg-1 melamine 30 days, there were slight damages to kidney,and calculus of the bladder appeared on male SD rats. The group whose ratio of established models was 70%with dosage of 0.3g·kg-1 was the highest.4.4 Melamine mainly induced urinary system damage including lesions of kidney tissues, calculus of the bladder and ureteral calculus and also slight damages to liver,spleen and lung.4.5 The Chinese Medicine Fu Fang San Jin Tang could get rid of melamine in mice and the preventive use of higher dose group had best effect.
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