Integrating Network Pharmacology And Animal Experiment To Explore The Mechanism Of Puerarin Improves Oliguria In Rats With Acute Alcoholism

Objective:By establishing the rat model of acute alcoholism,the mechanism of oliguria and puerarin treatment in acute alcoholism rats was investigated by combining network pharmacology with animal experiments.Methods:Puerarin-related targets were searched in the TCMSP,Pub Chem,CNKI,Wanfang,Pub Med and Geen Medical Academic databases.Potential acute alcoholism targets were obtained from the Gene Cards,Mala Cards and NCBI-gene databases.The puerarin-acute alcoholism intersection targets were obtained by Venny 2.1.0 online software.The interaction network for co-predicted targets was obtained using the STRING database,and core targets were imported into Cytoscape for visualization using DAVID Bioinformatics Resources 6.8.The essential genes were subjected to the Kyoto Encyclopedia of Genes and Genomes（KEGG）and Gene Ontology（GO）pathway enrichment analyses to predict related biological processes and significant signaling pathways.Finally,the binding ability of puerarin to the core targets was verified by molecular docking using Auto Dock software.The 48 male SD rats were randomly divided into four groups（n=12/group）,as follows:（1）Control（n=12）,（2）Ethanol（model,n=12）,（3）Ethanol+Puerarin（treatment,n=12）,（4）Puerarin（n=12）.A rat model of acute alcoholism was established through a one-time gavage of 56°white wine.In the comparison of urine volume changes in rats in each group,the morphological changes of kidney and hypothalamus tissues were observed by H&E staining;kits measured the plasma CRE and BUN concentrations;an automatic biochemical instrument to detect plasma Na⁺and urine Na⁺concentrations;ELISA was used to measure plasma copeptin concentration in rats;ADH,c AMP,PKA,CREB,c-Fos and CREB3L1 m RNA levels in hypothalamus tissues were detected by RT-q PCR;the distribution localization and protein expression of AQP₂ in renal tissues was discovered using immunohistochemistry;Western blot was used to measure the protein expression of AQP₂ in renal tissues and c AMP,PKA,CREB,p-CREB,c-Fos,ATF3 and CREB3L1in hypothalamic tissues.Results:1.The urine volume of rats in the model group was dramatically reduced compared to that of the rats in the control group（P<0.01）,and the urine volume of rats in the treatment group was increased compared to the model group（P<0.01）.2.Kidney and hypothalamus histopathological observations results:（1）There was no difference in the morphological structures of the glomeruli and tubules of the rats in each group were observed under light microscopy;（2）The number of hypothalamic tissue neurons decreased,cell bodies and nuclei grew larger and cell edema increased in the model group compared to the control group;compared with the model group,the hypothalamic tissue cell morphology of the treatment group was significantly improved.3.Compared with the control group,plasma CRE and Na⁺concentrations of rats in the model group had no significant changes,BUN concentration was significantly decreased（P<0.05）,and copeptin concentration was dramatically increased（P<0.05）.The treatment group copeptin concentration was substantially lower than the model group（P<0.05）.The results of RT-q PCR and Western blot showed that the expression levels of ADH m RNA in the hypothalamus and AQP₂ protein in the kidney were increased in the model group（P<0.05）.In contrast,ADH m RNA and AQP₂ protein expression levels were decreased in the treatment group（P<0.05）.According to immunohistochemical findings,compared with the control group,the expression of the cell membrane and cytoplasmic AQP₂ protein in kidney tissue was increased in the model group（P<0.05）;compared with the model group,the expression of the cell membrane and cytoplasmic AQP₂ protein in kidney tissue was significantly decreased in the treatment group（P<0.05）.4.A total of 214 intersectional targets were screened for puerarin and acute alcoholism by network pharmacology,and PPI network analysis identified 19 key targets.GO function and KEGG pathway enrichment analysis showed that the intersectional targets involved 837 biological processes and 185 signaling pathways.Molecular docking of the main targets and pathways showed that puerarin had good binding energy to c AMP,PKA,CREB and c-Fos.The results of RT-q PCR showed that the expression levels of c AMP,PKA,CREB,c-Fos and CREB3L1 m RNA in the hypothalamic tissues of the model group were significantly higher than that of the control group（P<0.05）;in contrast,the expression of c AMP,PKA,CREB,c-Fos and CREB3L1 m RNA in the hypothalamic tissues of the treatment group were significantly lower than that of the model group（P<0.05）.Western blot results showed that the protein expressions of c AMP,PKA,CREB,p-CREB,c-Fos,ATF3 and CREB3L1 in the hypothalamus of the model group increased when compared with the control group（P<0.05）;but compared with the model group,the protein expression levels of c AMP,PKA,CREB,p-CREB,c-Fos,ATF3 and CREB3L1in the hypothalamus of the treatment group were decreased（P<0.05）.Conclusion:1.Acute alcoholism leads to reduced urine output in rats,which was associated with upregulation of ADH and AQP₂ expression.2.Acute alcoholism causes an increase in ADH secretion associated with changes in c AMP,PKA,CREB,c-Fos,ATF3 and CREB3L1 proteins.3.Puerarin improves oliguria in rats with acute alcoholism by down-regulating the expression of c AMP,PKA,CREB,c-Fos,ATF3 and CREB3L1 proteins.