| ObjectIn this study,the calcium oxalate stone model rats were used as the research object to clarify the effect of Bushen Huashi Decoction on the formation of kidney stones;With the help of network pharmacology method,the key genes of Bushen Huashi Decoction in the treatment of renal calculi were obtained,and its possible mechanism of action was predicted.Furthermore,the expression of key genes and proteins in kidney tissue of calcium oxalate stone model rats was further verified by in vivo experiments,and the key proteins in relevant signaling pathways were detected for further verification,so as to systematically explain the mechanism of Bushen Huashi Decoction on the formation of kidney stones,and provide molecular biological basis for the clinical application of Bushen Huashi Decoction.Methods1.The model of calcium oxalate stone rats was reproduced by free drinking water of0.75% glycol and 0.75% ammonium chloride aqueous solution for 14 days.The model was divided into two ways: the dosage in the model making and the way of early administration.The therapeutic and preventive effects of bushengfossil Decoction on renal stones were determined by using 24 h urine volume,renal index,biochemical indexes of hematuria,oxidative stress level of renal tissue,oxalic acid and calcium level of renal tissue,apoptosis level of renal tubular epithelial cells and deposition of renal crystal.2.Mechanism prediction first,the target of active compounds and compounds of eight Chinese herbs of bushengfossil decoction was obtained by tcmsp database and literature retrieval.Meanwhile,the disease targets related to renal stones were screened by OMIM and genecards database,and drug targets and disease targets were analyzed to obegenes of drugs and diseases.Then,the PPI network is constructed by using string database,and the PPI network is analyzed by using the software of cyberscape 3.7.2.The core intersection genes are screened according to degree value.Finally,the core intersection gene was analyzed by means of David6.8 online analysis tool,and the results of the enrichment analysis were visualized by omicshare online analysis tool.3.The rat model of calcium oxalate calculus was established in the experience syndrome,and the key genes of estrogen signaling pathway in renal tissue,such as estrogen receptor 1(ESR1),estrogen receptor 1(ESR2),nuclear factor E2 related factor 2(Nrf2),heme oxygenase-1(HO-1),were detected by qPCR The m RNA expressions of B-cell lymphoma / leukemia 2(BCL2)and B-cell lymphoma2-associated X protein(Bax)were detected;Serum estradiol(17 β-Estradiol(E2)level;Western blot was used to detect the protein expression of ESR1,ESR2,Nrf2,HO-1,BCL2,and Bax in renal tissue,so as to elucidate the potential mechanism of Bushen Huashi Decoction in the treatment of renal calculi.Results1.Bushen Huashi Decoction has significant therapeutic and preventive effects on kidney stonesCompared with the normal group,the 24 h urine volume of the model group was significantly reduced;Renal index,serum creatinine,urea nitrogen,calcium and phosphorus levels,urinary oxalate and calcium levels,renal oxalate,calcium and malondialdehyde(MDA)levels were significantly increased,while serum magnesium level and renal total superoxide dismutase(SOD)activity were significantly decreased;He staining and von Kossa staining showed that the deposition area of renal crystal was significantly increased,and the renal tubules were dilated and the cells were edematous and exfoliated;TUNEL staining showed that the apoptosis index of renal tubular epithelial cells increased significantly.Compared with the model group,the 24-hour urine volume in the Bushen Huashi Decoction group was significantly increased;In addition,renal index,serum creatinine,urea nitrogen,calcium and phosphorus levels,urinary oxalate and calcium levels were significantly decreased,serum magnesium levels were significantly increased,renal oxalate,calcium and malondialdehyde levels were significantly decreased,while renal SOD levels were significantly increased;He staining and von Kossa staining showed that the kidney crystal deposition area was significantly reduced,renal tubular dilatation,cell edema and shedding were significantly reduced in the Bushen Huashi Decoction group;TUNEL staining showed that the apoptotic cell index of renal tubular epithelium decreased significantly.2.Network pharmacology predicts the mechanism of Bushen Huashi Decoction on the formation of kidney stonesAccording to the network pharmacology prediction,244 targets of Bushen Huashi Decoction and 1970 targets of kidney stones were obtained.The PPI network was constructed by taking the intersection genes of Bushen Huashi Decoction and kidney stones.The topological analysis of the PPI network was carried out by using Cytoscape 3.7.2 software.The nodes with the degree value more than 2 times of the median were taken as the core intersection genes,and 23 core intersection genes were obtained.The results of go functional enrichment analysis(top20)mainly involved the response to estradiol and the regulation of apoptosis;KEGG pathway enrichment analysis mainly involves nod like receptor signaling pathway,HIF-1signaling pathway and estrogen signaling pathway.Referring to the enrichment results of go and KEGG and literature research,it is speculated that Bushen Shihui decoction may play an anti kidney stone role by regulating estrogen signaling pathway.3.Verify the network pharmacology prediction pathway and key genes in vivo Compared with the normal group,the serum E2 level of the model group was significantly decreased by ELISA;However,Bushen Huashi Decoction can significantly increase the serum E2 level of rats with kidney stones.Compared with the normal group,the m RNA levels of ESR1,ESR2,BCL2,Nrf2 and HO-1 in the model group were significantly down regulated,while the m RNA levels of Bax were significantly up-regulated;Western blot results showed that the protein levels of ESR1,ESR2,BCL2,HO-1 and Nrf2 in renal tissue and nucleus of model group were significantly down regulated,while the protein level of Bax was significantly up-regulated;After treatment with Bushen Huashi Decoction,the abnormal expression of related genes and proteins in renal tissue of rats with renal calculi was significantly reversed.ConclusionBushen Huashi Decoction plays an anti-kidney stone formation effect by regulating the estrogen signaling pathway,improving the level of oxidative stress in the kidney and the apoptosis rate of renal tubular epithelial cells. |
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