| The incidence of chronic kidney disease(CKD)is increasing year by year in my country and the world.This problem increases the mental and economic burden of many patients and also poses a huge problem for public health.CKD refers to diseases with abnormal kidney structure or function>3 months,and its diagnostic criteria include:(1)albuminuria[AER≥30 mg/24 h;ACR≥30 mg/g(or≥3 mg/mmol)];(2)abnormal urine sediment;(3)renal tubule-related lesions;(4)histological abnormality;(5)structural abnormalities seen by imaging;(6)history of renal transplantation,glomerular filtration rate(GFR)decreased[eGFR<60 mL/(min·1.73m2)],the abnormal duration of any index can be diagnosed after more than 3 months.The Lancet Magazine published in 2012 that China’s epidemiological survey on CKD showed that the prevalence of CKD in adults is 10.8%,and it is estimated that 120 million adult patients with chronic kidney disease have an awareness rate of only 12.5%.The elements of syndrome of kidney-yang deficiency include Wen Xu’s dereliction of duty,waist house infertility,medullary emptiness,hyporeproduction,water flooding,yang deficiency and flooding,impetuous upset,impulsive and unregulated characteristics.Chinese medicine believes that chronic kidney disease can be attributed to the categories of "edema","guang","back pain" and other syndromes,and in the clinical diagnosis and treatment of the deficiency of the disease and the treatment of prescriptions and kidney yang deficiency syndrome is closely related.Based on this,modern Chinese medicine workers have conducted more clinical studies on the internal relationship between the two,such as the correspondence between the pathological changes of chronic kidney disease and the dialectical stages of traditional Chinese medicine,and the distribution of traditional Chinese medicine syndromes in chronic kidney disease.Therefore,based on the standardized elements of clinical TCM syndromes,it is of great significance to construct a rat model of chronic kidney disease and kidney-yang deficiency syndrome as a basic experimental research medium,and to screen and evaluate related models.In this study,male SD rats were used as experimental subjects.Adriamycin hydrochloride was injected into the tail vein,combined with hydroxyurea drug factors to induce rat modelling,and Zhenwu Tang was used to intervene drug counter evidence,and its macro-behavioral and biological indicators Test,and then make a more objective evaluation of this type of model and Zhenwu Tang to carry out a certain research and explain the possible intervention mechanism of this type of model.Object:1.To explore the specific method of establishing adriamycin combined with hydroxyurea to induce animal models of chronic kidney disease with kidney-yang deficiency syndrome,and to provide a more standard animal model of traditional Chinese medicine syndrome for the experimental and pharmaceutical research of chronic kidney disease with kidney-yang deficiency syndrome.2.Exploring and summarizing the evaluation system of the rat model of chronic kidney disease and kidney-yang deficiency syndrome,which is consistent with the theory of traditional Chinese medicine,including symptoms and signs,establishment of objective indicators,drug counter-evidence,etc.,and explaining the possible intervention mechanism of this model in Zhenwu Tang for chronic kidney The systematic study on the standardization of syndrome differentiation of diseased kidney-yang deficiency lays a certain biological foundation.Method:1.Establishment of rat model of chronic kidney disease with "Kidney Yang deficiency"syndromeCKD rat model was established by tail vein injection of adriamycin hydrochloride,and compound hydroxyurea was intragastrically administered to construct CKD rat model of"Kidney Yang deficiency" syndrome.Sixty-four male SD rats aged 6 to 8 weeks of SPF grade were fed with common feed.Forty-eight rats were selected to receive doxorubicin(3mg/kg,4mg/kg)via tail vein injection twice every other week,and 24h urine protein≥50mg was detected in rats,which means that the model of induced chronic kidney disease was successful.Based on urine protein and body weight,SD male rats successfully modeled were divided into normal group,doxorubicin group,doxorubicin compound hydroxyurea group,and doxorubicin compound hydroxyurea Zhenwu decoction group by stratified random method.Gavage.The physical state of the experimental rats was recorded,the body weight was weighed,the urine of the rats was collected for 24 hours,and the urine protein content of 24 hours was detected.Rats in each group were bled at the femoral artery at week 26,and various indexes were measured after serum was separated.The kidney,spleen and testis of the rat were weighed,and the kidney weight/weight ratio,spleen and testis index were calculated.2.Kidney paraffin embedding and sectioning,using HE and Masson staining to analyze the pathological changes of rat kidney;3.Colorimetric method:detect the content of AST,ALT,UA,UREA,TP,ALB,TC,TG and SOD in rat serum.4.Enzyme Linked Immunoassay:Detect the levels of T3,T4,TSH,FSH,LH,T,E2 in rat serum.5.The experimental data was statistically analyzed using SPSS 23.0.Result:1.Establishment and evaluation of CKD rat modelAfter induction by adriamycin,SD rats gradually developed lower limb edema,dull coat color and low gloss on the 4th week of the experiment;24h urine protein of the model group was higher than that of the normal group from the 5th week of the experiment(P<0,05);The 24h urine protein quantitative of SD rats in the model group was significantly higher than that in the normal group at 7,9,and 11 weeks(P<0.01),and≥50 mg was confirmed as a successful model.2.Establishment and evaluation of CKD kidney-yang deficiency rat model2.1 General situationAround the 12th week of the experiment,the rats developed symptoms of yang deficiency,such as hair loss,arched back,laziness,and curling up.Some rats were thinner and slower.2.2 24h urine protein quantificationIn the 12th week of the experiment,the 24h urine protein content of CKD kidney-yang deficiency rats showed that the urine protein of adriamycin and adriamycin hydroxyurea group was significantly higher than that of the normal group(P<0.01);the adriamycin hydroxyurea group was significantly It was higher than that of Zhenwu Decoction group(P<0.01).At the 16th,20th,and 24th week of the experiment,the quantitative statistical results of 24h urine protein in CKD kidney-yang deficiency rats showed that the trend of each group was similar to that at 12 weeks.2.3 Changes in body massAfter compound factors act on rats,their body weight shows a certain trend of change.At the 12th week of the experiment,the body weight of the normal group was significantly higher than that of the adriamycin group and the adriamycin hydroxyurea group(P<0.01);the weight of the Zhenwutang group was higher than the adriamycin hydroxyurea group(P<0.05).In the 15th week of the experiment,the weight of the normal group was significantly higher than that of the adriamycin and adriamycin hydroxyurea group(P<0.01);the 18th,21st,23rd,and 26th weeks of the experiment were similar to the previous experimental nodes,and the weight of the normal group was significantly higher Adriamycin,adriamycin hydroxyurea group and Zhenwu decoction group(P<0.01).2.4 Swimming experimentOn the 16th week of the experiment,the average swimming time of rats in the normal group was the longest,which was significantly higher than that of the adriamycin group and the adriamycin hydroxyurea group(P<0.01);Similar for 16 weeks.2.5 Infrared temperature measurementInfrared temperature measurement is a certain digital temperature after the infrared radiation of the animal body surface is sensed by infrared.Infrared thermal imaging receives the infrared rays radiated from the animal’s body surface through an infrared collection lens,and marks different temperature levels from low to high temperatures,and then displays the invisible heat field information and distribution on the animal’s surface in an image visible to the human eye.Qualitative evaluation is made by observing and analyzing the infrared radiation trajectory represented by the color scale distribution of the collected objects.In recent years,there have been more studies and applications of these two technologies to judge the degree of yang deficiency in animal models.After temperature measurement,the statistical results of left forepaw temperature of CKD kidney-yang deficiency rats showed that the difference between the adriamycin hydroxyurea group and the Zhenwu decoction group was not significant,but there was a certain trend at the average value level.The statistical results of the tail root temperature of rats with CKD kidney-yang deficiency showed that the temperature of the normal group was significantly higher than that of the adriamycin and adriamycin hydroxyurea groups(P<0.01).The nose tip,upper and lower limbs,tail roots and left and right kidney projection areas were selected as infrared thermal imaging detection sites.In the nose tip,the temperature of the adriamycin group was significantly higher than that of the adriamycin hydroxyurea group(P<0.01);in the left upper limb,The temperature of the normal group was significantly higher than that of the adriamycin hydroxyurea group(P<0.01);the left lower limb,right upper limb,and right lower limb were not statistically significant;the tail root and left and right kidney projection areas were not statistically significant in each group significance.2.6 Kidney pathological changes in CKD rats with kidney-yang deficiencyThe results of HE and Masson staining of rat kidney tissues showed that the rats in the adriamycin and adriamycin hydroxyurea group were wider in the glomerular mesangial area than the normal group,and the mesangial cells showed different degrees of proliferation,and the kidneys were affected by the disease.Some renal tubular epithelial cells in the glomerulus have vacuolar changes,and a large number of inflammatory cells infiltrate the renal interstitium.In the Zhenwu Decoction intervention group,glomerular mesangial cells were slightly proliferated,and there was a small amount of inflammatory cell infiltration in the renal interstitium.3.Study on the intervention mechanism of Zhenwu Decoction on CKD kidney-yang deficiency rat model3.1 Kidney weight to weight ratio and organ indexThe average level of kidney weight to weight ratio of rats in the adriamycin group was higher than that of the normal group after the 26th week of the experiment;the kidney weight ratio of rats in the adriamycin hydroxyurea group was higher than that of the normal group(P<0.05).There was no statistical difference in the spleen index between the groups;the appearance of the testes of rats in the adriamycin hydroxyurea group and the Zhenwu decoction group was significantly smaller than that of the normal group and the adriamycin group,and compared with the normal group and the adriamycin group,The testicular index of rats was significantly reduced(P<0.01).3.2 Serological indicators1)Liver functionAST:Compared with the normal group,the adriamycin group,and the adriamycin hydroxyurea group,the statistical difference is not significant,but the average level of each group has a certain trend;ALT:The adriamycin group was significantly higher than the normal group and Zhenwu decoction group(P<0.01).2)Kidney functionScr:There is no statistical difference between the groups;UA:Compared with the normal group,the Zhenwu Decoction group has a statistical difference(P<0.05);BUN:Zhenwutang group was significantly lower than adriamycin group(P<0.01).3)Blood lipids and albuminTC:There was no statistical difference between the groups,but the average level of the adriamycin group,adriamycin hydroxyurea group,and Zhenwu decoction group was higher than that of the normal group,and the level of Zhenwu decoction group was lower than that of the adriamycin hydroxyurea group.TG:The adriamycin group was higher than the normal group(P<0.05),and the average values of the adriamycin hydroxyurea group and the Zhenwu decoction group were higher than the normal group.TP、ALB:the average values of adriamycin group,adriamycin hydroxyurea group,and Zhenwu decoction group were lower than those of the normal group,and the Zhenwu decoction group was significantly different from the normal group(P<0.05).4)Thyroid hormoneT3:Adriamycin hydroxyurea group and Zhenwu decoction group were significantly lower than normal group(P<0.01),adriamycin hydroxyurea group and Zhenwu decoction group were significantly lower than adriamycin group(P<0.01);T4:Adriamycin group,adriamycin hydroxyurea group,Zhenwu decoction group were significantly lower than the normal group(P<0.01);Zhenwutang group was significantly higher than adriamycin hydroxyurea group(P<0.01);TSH:Adriamycin group,adriamycin hydroxyurea group,Zhenwu decoction group were significantly higher than the normal group(P<0.01);Zhenwu decoction group was significantly higher than adriamycin hydroxyurea group(P<0.01).5)Gonadal hormonesT:The average level of each group has a trend,the normal group is higher than the adriamycin hydroxyurea group,the Zhenwu decoction group,and the Zhenwu decoction group is higher than the adriamycin hydroxyurea group;E2:The level of adriamycin group was significantly lower than that of the normal group(P<0.01);the level of adriamycin hydroxyurea group was lower than that of the normal group(P<0.05);the level of adriamycin hydroxyurea group was higher than that of the adriamycin group(P<0.01);The level of Zhenwu Decoction group was significantly higher than that of Adriamycin group(P<0.01);FSH:The levels of adriamycin group,adriamycin hydroxyurea group,and Zhenwu decoction group were significantly lower than those of the normal group(P<0.01);Zhenwu decoction group was significantly higher than that of adriamycin hydroxyurea group(P<0.05);LH:The levels of adriamycin group,adriamycin hydroxyurea group,and Zhenwu decoction group were significantly lower than those of the normal group(P<0.01);the level of Zhenwu decoction group was significantly higher than that of the adriamycin group(P<0.01).6)Superoxide dismutaseCompared with the normal group,the adriamycin group,adriamycin hydroxyurea group,and Zhenwu decoction group have significant differences(P<0.01);the Zhenwu decoction group is significantly higher than the adriamycin group(P<0.01);Zhenwu The soup group was significantly higher than the adriamycin hydroxyurea group(P<0.01).7)cAmp,cGmpcAmp:The level of adriamycin hydroxyurea group was significantly higher than that of the normal group(P<0.01);the Zhenwutang group was higher than that of the normal group(P<0.05),and there was no difference among the other groups;cGmp:The adriamycin hydroxyurea group was higher than the normal group(P<0.05);the Zhenwu decoction group was higher than the adriamycin group(P<0.05),and there was no difference among the other groups;cAmp/cGmp:There is no statistical difference between the groups.Conclusion:1.By using the tail vein to inject doxorubicin hydrochloride(3mg/kg,4mg/kg)combined with hydroxyurea(375mg/kg)doses can successfully induce CKD kidney-yang deficiency model in rats;2.Detect the stability of the CKD kidney-yang deficiency rat model through various evaluation experiments of the model’s general condition,endurance change,and metabolic capacity.The performance of the model group of rats is more consistent with the main symptoms in clinical differentiation.Whether the establishment of the induction model has corroborative effect;3.In the model group,the thyroid and gonads and other organ functions of rats in the model group are in a state of inhibition.Zhenwu Decoction is used as both the drug counter-syndrome group and the intervention factor.The results show that the Zhenwu Decoction group performs better than the model group.It shows that Zhenwu Decoction has a therapeutic effect on the rat model of kidney-yang deficiency induced by adriamycin combined with hydroxyurea,and also supports the scientific feasibility of this model. |
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