Mechanistic study of Shilinqing Granules intervening in PI3K/Akt/mTOR pathway for relieving autophagy-induced calcium oxalate kidney stones in rats
OBJECTIVE To explore the mechanism of Shilinqing Granules(SG)in ameliorating calcium oxalate(CaOx)renal stones through network pharmacology,molecular docking and experimental verification in rats.METHODS Drug compo-nents and targets were screened by the database of Traditional Chinese Medicine Systems Pharmacology(TCMSP)And disease targets were acquired through the databases of GeneCards,OMIM and TTD.The intersection of component-disease targets was examined by Venny software.Cytoscape 3.9.0 software was employed for constructing a network diagram of"SG-active components-targets-disease".Protein-protein interaction(PPI)network analysis of functional targets was performed using the String database and GO and KEGG pathway enrichment analysis was conducted with Bioconductor software.Molecular docking was performed with Autodock Tools.Rat model of CaOx renal stones was established with 1％ethylene glycol and 2％ammo-nium chloride.The serum levels of blood urea nitrogen(BUN),creatinine(Cr)and calcium(Ca2+)were determined;the patho-logical changes of kidney,crystallization and autophagy in renal tubular epithelial cells were observed;Western blot was utilized for detecting the protein expression levels of Beclin-1,LC3 Ⅱ/Ⅰ,p62,p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR.RESULTS A total of 189 drug targets of SG,3 367 targets of kidney stones and the intersection of 130 targets were obtained.The core targets included Akt,interleukin-1β(IL-1 β),interleukin-6(IL-6)and tumor necrosis factor(TNF).The major signal-ing pathways involved were PI3K/Akt,interleukin-17(IL-17)and TNF.Molecular docking indicated that the active components had excellent binding activity with core targets.The experimental results showed that the serum levels of BUN,Cr and Ca2+declined(P＜0.05 or P＜0.01);calcium salt deposition in renal tubules and mitochondrial cristae fractures and dissolution in renal tubular epithelial cells,as well as the number of autophagic lysosome dropped obviously;the protein expressions of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR spiked markedly(P＜0.01),ratio of Beclin-1 and LC3 Ⅱ/Ⅰ dipped obviously(P＜0.01)and the expression of p62 was significantly up-regulated(P＜0.01).CONCLUSION SG may effectively improve the level of autophagic stress in renal tissue of rats,thereby suppressing the formation of CaOx kidney stones.Its mechanism of action is probably correlated with a up-regulation of PI3K/Akt/mTOR signaling pathway.
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