Analysis Of The Mechanism Of Alismatis Rhizoma Processed With Salt In Improving The Edema Model Of Kidney Yin Deficiency In Rats

ObjectiveTo establish the rat model with edema of kidney yin deficiency（EKYD）induced by tail intravenous injection of doxorubicin combined with intragastric administration of thyroid hormone tablets,to observe the difference of pharmacological effects of Alismatis Rhizome（AR）and salt-processed AR（SAR）on the prevention and treatment of EKYD,and to explore its mechanism.The differences in components in vivo and in vitro were compared between AR and SAR,and the relationship between the efficacy difference and components was preliminarily expounded.Method1.Efficacy evaluation of AR and SAR on EKYD:Male SD rats were randomly divided into a normal group,model group(thyroxine 75 mg kg^-1+adriamycin 2 mg kg^-1 d^-1),and positive drug group[Liuwei Dihuang pills,1.4 g(kg^-1 d^-1)]after adaptive feeding.There were 6 rats in each group,including low-dose AR group[1 g(kg^-1·d^-1)],high-dose AR group[4 g(kg^-1·d^-1)],low-dose SAR group[1 g(kg^-1·d^-1)],high-dose SAR group[4 g(kg^-1·d^-1)].The rats in the normal group and the model group were given the same volume of drinking water by stomach on the first day,and the other groups were given the corresponding drugs by stomach for 28 days.The volume was 1 m L(100 g^-1d^-1).The rats in each group were placed in metabolic cages immediately after 14 days and the last day of drug administration,and 24 hours later,the amount of drinking water and urine of each cage rats were recorded,and an appropriate amount of urine of each cage rat was retained.The contents of urinary protein,urinary creatinine and urinary albumin were measured by automatic biochemical analyzer,and the concentrations of sodium,potassium,and chloride in urine on the last day were detected.The contents of CREA,UREA,ALB,TP,CHOL,and TG in serum were determined by the automatic biochemical analyzer.The pathological changes in the kidney were observed by HE staining,the expression of AQP-1,AQP-2,NHE3,andγ-ENa C protein in the kidney was detected by immunohistochemical method,and the transcription level of AQP-1and AQP-2 m RNA in kidney was determined by Real-time PCR.2.Study on the mechanism of prevention and treatment of EKYD before and after treatment with AR and SAR:Based on the Illumina Hi Seq X sequencing platform,transcriptome sequencing analysis was carried out on the kidney tissue samples of normal group,model group,low-dose AR group and low-dose SAR group.By analyzing the difference in gene expression in the kidney of rats in each group,the differential gene set was constructed and enriched by GO and KEGG.At the same time,the results of AR and SAR were compared,the key signal pathways were screened,and the interaction of key genes shared by AR and SAR was analyzed.The core targets were selected to explore the potential mechanism of AR and its processed products in improving the EKYD from the molecular level and to explore the potential reasons for the synergistic effect of SAR.3.Study on the changes in the chemical composition of AR and SAR:（1）The difference in the chemical composition of AR before and after preparation was analyzed in vitro.The chemical composition of AR before and after processing was determined by UPLC-Q-TOF-MS,and combined with SIMCA 14.1 software analysis the differences in chemical composition between AR and SAR.（2）The serum pharmacochemistry of AR and SAR were compared and analyzed.After adaptive feeding,SD male rats were randomly divided into a normal group（n=3）,AR group（n=3）,and SAR group（n=3）.The corresponding drug extract was given by intragastric administration of 10 g kg^-1（10 times the bioequivalent dose of rats）,once in the morning and evening for 3consecutive days.The blank group was given the same volume of drinking water,and the orbital blood samples were collected after the last administration of 60 min.The supernatant was collected by centrifugation,and the serum samples of the same group were mixed,then the protein was precipitated by acetonitrile.After centrifugal concentration,the supernatant was re-dissolved with acetonitrile,and the total ion current map of each sample was detected by UPLC-Q-TOF-MS,and the data were analyzed and processed by UNIFI software.Results1.Pharmacodynamic evaluation of AR and SAR on EKYD model rats:The results showed that AR and SAR could improve renal filtration rate,T3,T4,c AMP,c GMP,and lipid metabolism,and the related evaluation indexes were adjusted.It is speculated that its mechanism may be related to correcting the disorder of the HPG axis and inhibiting the expression of water and sodium channel proteins related to AQP-1 and AQP-2.And in the HPG axis,renal filtration rate,the degree of yin deficiency and water and sodium channels and other indicators,it was found that the effect of the low-dose SAR group was slightly better than that of the low-dose AR group.2.Study on the mechanism of prevention and treatment of EKYD model rats with AR and SAR:Compared with the model group,there were 522 and 682 differential genes in AR and SAR low-dose groups,respectively.The expression of 352 and 480differential genes were significantly adjusted after administration（P<0.05）.Enrichment analysis showed that both AR and SAR low-dose groups could regulate related inflammatory response and immune processes,and the number of gene targets regulated by the low-dose SAR group was much more than that in the low-dose AR group,and both of them were significantly enriched in PI3K-Akt signaling pathway.It was speculated that may be the main pathway for AR and SAR to improve EKYD.Ten core targets such as PTPRC,CD4,and LCP2 were screened out by the interaction of the PPI protein network.3.Study on the changes in the chemical composition of AR and SAR:（1）The chemical composition of AR before and after processing was determined by UPLC-Q-TOF-MS.It was found that the change in terpenoid composition before and after processing was not obvious,and the increased or decreased chemical components were not found in the experiment.Taking the peak area of each compound as a variable for multivariate statistical analysis,the results showed that there was a significant change in the compound content,showing a cluster distribution,indicating that there was a significant change in the content of AR before and after preparation.（2）The differences in blood components of AR and SAR were compared by serum pharmacochemical analysis.It was found that 20 and 14 components were inferred in the serum of rats treated with water extract of AR and SAR,respectively,of which 13 components were in common,including 5 prototype components and 8 metabolic components.It is speculated that the possible material basis of the therapeutic effect of AR and SAR were 16-oxo-alisol A,alisol A 24-acetate,alisol B-23-acetate,alisol C-23-acetate and alisol A,B,C.Conclusion1.AR has a therapeutic effect on EKYD model rats before and after preparation,and the low-dose SAR group is better than the low dose AR group.2.AR and SAR may improve the EKYD by regulating the PI3K-Akt signaling pathway,and the number of regulatory targets in the low-dose SAR group is more than that in the low-dose AR group,and the function is more extensive,which may be the potential reason why the efficacy of the low-dose SAR group is better than that of the low-dose AR group.3.In the process of AR salt preparation,it had little effect on the types of chemical components,mainly changed the content of compounds,but there was little difference between the two blood components,and the metabolites of AR were more abundant than that of SAR.Salt preparation increases the content of some blood components.