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Objective::To explore the active components, potential targets and signaling pathways of Rhei Radix et Rhizoma in the treatment of renal fibrosis based on the network pharmacology method, and then to verify the target genes in vitro. Method::Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID) were retrieved to obtain the main active ingredients of Rhei Radix et Rhizoma. The potential anti-renal fibrosis targets of Rhei Radix et Rhizoma were predicted by similarity ensemble approach (SEA), Swiss Institute of Bioinformatics (SIB) and GeneCards Database. Target protein-protein interaction (PPI) network was constructed by using String Version 10.5 database. David 6.8 software was used for gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of the key targets. Cytoscape Version 3.6.0 software was used for visualized analysis of PPI network, active ingredient-key target network and the ingredient-target-signal pathway network. In combination with Malachards database, the signal pathways with high correlation with renal fibrosis were screened. Then, cell experiments were used for verification: HK-2 cells were selected to establish fibrosis model by transforming growth factor-β1 (TGF-β1) stimulation. The cells were treated with rhein for 48 hours. Western blot assay was used to detect the protein expression level of hypoxia inducible factor-1 α (HIF-1 α), vascular endothelial growth factor (VEGF), and platelet-derived growth factor receptor-α (PDGFR-α). Protein expression levels of E-cadherin and α smooth muscle actin (α-SMA) were detected by immunofluorescence. Apoptosis was detected with flow cytometry. Result::Totally 17 active ingredients of Rhei Radix et Rhizoma and 424 targets of anti-renal fibrosis effect were screened out, including five key targets: protein kinase B(Akt)1, mitogen activated protein kinases 3(MAPK3), epidermal growth factor receptor(EGFR), interleukin(IL)-6 and VEGFA in turn. The biological process of GO enrichment mainly involved signal transduction, cell proliferation and apoptotic process. The results of KEGG pathway enrichment showed that phosphatidylinositol 3-kinase(PI3K)/Akt, HIF-1, VEGF, and forkhead transcription factor (FoxO) pathways were related to the anti-renal fibrosis mechanism of Rhei Radix et Rhizoma. Results of the in vitro experiment proved that rhein could inhibit the expression of E-cadherin, α-SMA, HIF-1α, VEGF and PDGFR-α. In addition, rhein inhibited apoptosis induced by TGF-β1 in HK-2 cells. Part of the prediction results of network pharmacology were verified. Conclusion::This study reflects the multi-component, multi-target and multi-pathway mechanism characteristics of Rhei Radix et Rhizoma. The mechanisms of its anti-renal fibrosis effects may be related to inhibiting HIF-1 α / VEGF /PDGFR-α signaling pathway, apoptosis and epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells.
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Objective:To explore the effect of different effective parts of Taohe Chengqitang on the synthesis and degradation of extracellular matrix in human kideny-2（HK-2） cells induced by transforming growth factor-β1（TGF-β1）. Method:Petroleum ether extract, ethyl acetate extract, n-butanol extract, raffinate and polysaccharide extract, mirabilite extract were extracted with 70% ethanol by systematic solvent method. The HK-2 cell fibrosis model induced by TGF-β1 was built to intervene the cells in different parts of Taohe Chengqitang with different concentrations (0, 50, 100, 200, 400, 800 mg·L-1). Enzyme-linked immunosorbent assay（ELISA）kit assay was used to detect collagen（Col）-Ⅰα1 and fibronectin （FN）in supernatant to screen out the main active parts. Cell counting kit-8 （CCK-8）method was used to determine the best concentration of intervention site of bioactive components. Western blot analysis was used to detect the expression levels of Col-Ⅰ, Col-Ⅲ, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase inhibitor2 (TIMP2), and connective tissue growth factor (CTGF). Immunofluorescence assay was used to detect the expression of α-smooth muscle actin（α-SMA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） analysis was used to detect the mRNA expression of plasminogen activator inhibitor-1（PAI-1）. Result:ELISA kit assay demonstrated that compared with the model group, ethyl acetate extract, n-butanol extract and chloroform extract significantly reduced the Col-Ⅰα1 and FN content at the concentrations of 200 and 400 mg·L-1 (P<0.05, P<0.01). CCK-8 assay showed that the cells viability was significantly inhibited with drug intervention at the concentrations of 400 and 800 mg·L-1 (P<0.01). Western blot demonstrated that compared with the model group, ethyl acetate extract, n-butanol extract and chloroform extract decreased the expression levels of Col-Ⅰ, Col-Ⅲ, TIMP2 and CTGF in HK-2 cells induced by TGF-β1, and increased the expression of MMP-2 (P<0.05), with more significant effect in n-butanol extract (P<0.01). The results of immunofluorescence showed that ethyl acetate extract, n-butanol extract and chloroform extract could inhibit the expression of α-SMA (P<0.05), with more significant effect in n-butanol extract (P<0.01). The results of Real-time PCR showed that ethyl acetate extract and chloroform extract inhibited mRNA expression of PAI-1 (P<0.05), with more significant effect in n-butanol extract (P<0.01). Conclusion:The extracts of ethyl acetate, n-butanol and chloroform are the active parts of Taohe Chengqitang with the anti-renal fibrosis effect, with n-butanol extract as the most active part. The mechanism on anti-renal fibrosis may be related to its regulation of extracellular matrix (ECM) synthesis and degradation.
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<p><b>OBJECTIVE</b>To establish a method of high-performance liquid chromatography (HPLC) for determining the urine oxalate levle in rats with renal calcium oxalate calculus.</p><p><b>METHODS</b>Totally 24 SPF Wistar healthy male rats were randomly divided into control group(n=12)and ethylene glycol (EG) group (n=12). Rats in EG group were administered intragastrically with 2% ammonium chloride (AC)2 ml/rat per day+1% ethylene glycol (EG), along with free access to drinking water.The control group was fed with deionized water, along with the intragastric administration of normal saline (1 ml per day). Twenty-eight days after modelling, the 24-hour urine samples were collected, and the urine oxalic acid levels were determined using HPLC and the results were compared with those of catalytic spectrophotometry using oxidation of methyl. During the HPLC, the samples were separated on Aglient 5TC-C18 (250×4.6 mm,5 Μm), eluted with mixture of methanol (0.1 mol/L) and ammonium acetate (15:85) at 1.2 ml/min, and detected at 314 nm, with the column temperature being 20 ℃.</p><p><b>RESULTS</b>The standard curves of high and low concentrations of oxalic acid were y=5909.1x+378730, R² =0.9984 and y=7810.5x-16635, R² =0.9967,respectively. The lowest detectable concentration in this method was 5 Μg/ml. The linear high concentration range of oxalate stood at 62.50-2000.00 Μg/ml, and the linear low concentration range of oxalate stood at 6.25-100.00 Μg/ml. Its average recovery was 95.1%, and its within-day and day-to-day precisions were 3.4%-10.8% and 3.8%-9.4%. Both HPLC and catalytic spectrophotometry showed significantly higher urinary oxalic acid concentration and 24 h urine oxalate level in EG group compared with the control group [urinary oxalic acid concentration: (736.35 ± 254.52) Μg/ml vs.(51.56 ± 36.34) Μg/ml,(687.35 ± 234.53) Μg/ml vs.(50.24 ± 42.34) Μg/ml;24 h urine oxalate level: (11.23 ± 4.12)mg vs.(0.87 ± 0.45)mg,(9.89 ± 3.55)mg vs. (0.77 ± 0.65)mg; all P<0.01]. No statistically significant difference was observed in the results of urinary oxalate concentration and 24 h urine oxalate level between HPLC and potassium chromate oxidation of methyl red spectrophotometry (all P>0.05).</p><p><b>CONCLUSION</b>HPLC is a simple, rapid, and precise method in detecting urine oxalate level in rats with renal calcium oxalate calculus, with high recovery rate.</p>