| Objective(一)The difference in toxicity of AAI was evaluated by neonatal mice and adult mice,which provided a scientific basis for the clinical dialectical use of AAI and its safe and rational application.(二)The metabolic differences of AAI and AAT in different age groups were evaluated by neonatal mice and adult mice,and the causes of aristolochic acid toxicity differences in different age groups were explained scientifically.(三)To evaluate the therapeutic effect of Huoxuehuayu formula based on Danggui Shaoyao San on liver and kidney toxicity in adult mice induced by AAI,and to provide a scientific and effective treatment for patients with aristolochic acid-related preparations.Methods(一)Study on the toxicity of AAI in neonatal mice and adult mice.The study was divided into the normal control group of neonatal mice,AAI 2.5mg /kg group of neonatal mice,AAI 5mg /kg group of neonatal mice,normal control group of adult mice,AAI 2.2mg /kg group of adult mice,and AAI 4.4mg /kg group of adult mice.15 mice in each group were given the AAI continuously for 5 days.The mortality of AAI-induced neonatal mice and adult mice was observed.Serum was collected for liver,kidney and heart function tests.Liver,kidney and heart tissue samples were collected for HE staining to compare the differences in organ injury.(二)Study on the difference in metabolism between AAI and AAT in neonatal and adult mice.The study was divided into the normal control group of neonatal mice,the AAI group of 22.9 mg/kg neonatal mice,the normal control group of adult mice,the AAI group of 20 mg/kg adult mice,and the intraperitoneal injection of AAI once.Blood was collected from the eyes at 2,4,6,8,10,12 and 24 hours after the beginning of the experiment,and urine was collected at the corresponding time points,three neonatal mice and adult mice in each group.The lc-ms/ms method was used to detect the differences in blood and urine contents of AAI and AAT in neonatal mice and adult mice,which further explained the reasons for the differences in toxicity of AAI in mice of different age groups,and provided the corresponding scientific basis for the clinical treatment of diseases induced by the use of AAI-related preparations.(三)To evaluate the therapeutic effect of Huoxuehuayu formula on on liver and kidney toxicity induced by AAI in adult mice.Adult mice were randomly divided into 4 groups: control group,model group,low-dose group of Huoxuehuayu formula,high-dose group,,10 mice in each group,model group were intraperitoneally injected with AAI 4.4mg /kg for 5 days,low-dose group,high-dose group were administrated with Huoxuehuayu formula of 7.5g /kg and 15 g /kg,respectively,for 14 days.The overall situation of the experimental animals was observed during the experiment.Serum was collected for liver and kidney function testing,liver and kidney tissue samples were collected for HE staining to compare the differences in organ injury.Results(一)Study on the toxicity of AAI in neonatal mice and adult mice.Death of experimental animals: death occurred on day 8 in the adult AAI group of 2.2 mg/kg,with the final mortality rate of 53.3%;death occurred on day 6 in the adult AAI group of 4.4 mg/kg,with the final mortality rate of 100%,while no death occurred in the neonatal mice group.Serological results showed that compared with the blank control group,adult mice creatinine(Cr),urea nitrogen(BUN),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate hydrogenase(LDH)caused by AAI were significantly increased(p < 0.05)in a dose-dependent manner.Among them,Cr and BUN increased most significantly,but it had no effect on the indicators of neonatal mice.HE staining results showed that adult mice could cause acute tubular necrosis after intraperitoneal injection of AAI.Most hepatocytes have nuclear lysis and nuclear fragmentation;myocardial congestion,partial myocardial fiber necrosis,the degree of above lesions are proportional to the dose of AAI;Among them,kidney damage is the most serious,liver and heart damage are relatively light,and AAI has no obvious damage to the kidney,liver and heart of neonatal mice.(二)Study on the difference in metabolism between AAI and AAT in neonatal and adult mice.The peak concentration was rapidly reached 2 h after the experiment.At the peak concentration,the concentration of AAI in the blood of neonatal mice was only 55.9% of that of adult mice,and then showed the same trend of decline.The change trend of AAI concentration in urine was consistent with that in blood;At peak concentration,there was no significant difference in the blood concentration of AAT in neonatal mice and adult mice,but there was a significant difference in the urine concentration of AAT in neonatal mice at peak concentration,which was only 35.1% of that in adult mice.(三)To evaluate the therapeutic effect of Huoxuehuayu formula on on liver and kidney toxicity induced by AAI in adult mice.The adult mice AAI 4.4 mg/kg group died on the 6th day,with a final mortality rate of 100%,while the Huoxue Huayu formula 7.5 g/kg and 15 g/kg groups died on the 8th and 10 th days,respectively.The final survival rate was 30% and 50%,respectively.In the experiment,their weight gradually increased,vitality increased,hair became smoother,and their activity was stronger.Serological test results showed that compared with the control group,the levels of creatinine(Cr),urea nitrogen(BUN),alanine aminotransferase(ALT),aspartate aminotransferase(AST)in model mice significantly increased(p <0.05).However,compared with the model group,the above indexes were significantly decreased in the Huoxue Huayu formula 15 g/kg group after treatment(p <0.05).HE staining results showed that compared with the control group,renal tubular necrosis was severe,lymphocytes infiltrated significantly,and hepatic lobes most hepatocytes had nuclear lysis and nuclear fragmentation,and a small amount of neutrophil and lymphocyte infiltration.Compared with the model group,the degree of renal tubular injury was significantly reduced in the Huoxue Huayu formula 15 g/kg group,and lymphocyte infiltration was reduced.Nuclear are lysed and broken,and inflammatory cell infiltration is reduced.ConclusionsIn summary,this topic evaluated the toxicity of AAI in neonatal and adult mice and the difference in metabolism in vivo.It was found that under the same volume specific dose and at the same time,the blood intake of AAI in adult mice is significantly higher than that in neonatal mice,which explains the reason for the difference in death between aristolochic acid I and neonatal and adult mice,and the metabolic capacity of neonatal mice to AAI is significantly higher than that of adult mice,but The excretion rate of AAT is significantly lower than that of adult mice,and AAT tends to accumulate in neonatal mice;The study found that Huoxue Huayu formula can reduce the toxicity of AAI in adult mice by improving liver and kidney functions,and provide a new treatment perspective and program for patients who mistakenly take aristolochic acid related preparations. |
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