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Objective @#To establish an in vitro renal injury model of amikacin (AKN) and investigate the protective effect and mechanism of vaccarin (VA) in the AKN-induced in vitro renal injury model .@*Methods @#Human renal tubular epithelial cells (HK-2) were cultured in vitro and incubated with different drugs of AKN or/and VA to de- termine the optimal drug concentration based on cell viability tested by MTT. The changes in intracellular oxidative stress were assessed using the dihydroethidium ( DHE) probe and malondialdehyde ( MDA) /glutathione ( GSH) assay kits at different time points . Total RNA was extracted , and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1 ( KIM-1) and neutropil gelatinase-associated lipocalin ( NGAL) . Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in HK-2 cell lysis .@*Results @#High concentrations of AKN significantly decreased the viability of HK-2 cells in vitro , with a half maximal inhibitory concentration (IC50) of (5 . 74 ±0. 47) mmol/L. VA at concentrations of 25 - 100 μmol/L increased the viability of AKN-stimulated HK- 2 cells (P < 0. 05) . After treatment with AKN (4 mmol/L) , the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control (NC) group ( P < 0. 001) . VA (50 μmol/L) significantly reduced the mRNA expression levels of KIM-1 (P < 0. 01) and NGAL (P < 0. 05) . The intensity of DHE staining increased after 3 hours of AKN treatment , but the difference was not statistically significant. However , the intensity of DHE staining was significantly higher in the 6 - 24 hours group compared to the 0 - hour group (P < 0. 01) . Furthermore , MDA levels significantly increased , while GSH levels significantly decreased after 6 - 24 hours of AKN treatment , with statistically significant differences (P < 0. 05) . After 6 - 24 hours of AKN stimula- tion , the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased (P < 0. 001) . Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining , MDA and GSH levels , as well as the chan- ges of SLC7A11 and GPX4 protein levels (P < 0. 001) .@*Conclusion @#In this study , an in vitro renal injury model was established by stimulating HK-2 cells with high concentrations of AKN , and it was found that VA might allevi- ate the damage to renal tubular cells caused by AKN via inhibiting oxidative stress related ferroptosis .
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Aim To investigate the effect of vaccarin on mouse atherosclerosis in vivo and the underlying mechanism. Methods AopE mice aged 6 to 8 weeks old were used to establish the atherosclerosis model. Oil red O staining was used to determine the lipid levels in aorta and aortic root. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory factors. Results Vaccarin could effectively reduce the levels of blood glucose and blood pressure in AopE
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OBJECTIVE：Compare the fingerprint difference of Vaccariae Semen before and after processed （stir-fried），and to determine the contents of erythrine and vaccarin before and after stir-fried. METHODS ：UPLC method was adopted. The determination was performed on YMC Trait C 18 column with mobile phase consisted of acetonitrile-water （gradient elution ）at the flow rate of 0.35 mL/min. The detection wavelength was set at 219 nm，and the column temperature was 35 ℃. The sample size was 1 μL. Using vaccarin as reference，the fingerprints of Vaccariae Semen crude product and its processed product （each of 17 batches，S1-S17，CS18-CS34） were drawn. The similarity evaluation and common peak identification were carried out by Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）；cluster analysis ，principle component analysis （PCA）and factor analysis were performed by using SPSS 20.0 software. The contents of erythrine and vaccarin in Vaccariae Semen crude product and its processed product were determined by UPLC. RESULTS ：There were 5 common peaks in UPLC fingerprints of 17 batches of Vaccariae Semen crude product and its processed product. The similarities were all higher than 0.99. Among them ， 2 common peaks were identified ，i.e. erythrine ，vaccarin. Results of cluster analysis showed that S 1-S17 were clustered into one category and CS 18-CS34 were clustered into one category. Results of PCA and factor analysis showed that variance contribution rate of the first principle component was 76.418％；erythrine and vaccarin had higher loading on the first principal component （eigenvalues were 0.976 and 0.966，respectively）. The linear ranges of above 2 components were 6.437-321.832 μg/mL and 7.729-386.437 μg/mL，respectively（r＞0.999）. The limits of detection and quantitation were 0.085，0.284 ng （crude product） and 0.739， 2.465 ng （processed product ）， respectively. RSDs of precision ，reproducibility，stability（12 h）and durability tests were all lower than 3％（n＝6 or n＝5）. E-mail：1083656123@qq.com Average recoveries were 96.42％（RSD＝0.85％，n＝6）and 99.13％（RSD＝1.74％，n＝6）. The contents of the two components were 0.11％-0.20％，0.42％-0.63％（crude product ）and 0.08％-0.11％，0.34％-0.50％（processed product ）. CONCLUSIONS ：UPLC fingerprint of Vaccariae Semen crude product and its processed product are established successfully. Although the chemical constituents in Vaccariae Semen are consistent before and after stir-fried ，the contents of erythrine and vaccarin are all decreased after stir-fried.
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Objective: To establish the culture system of Vaccaria segetalis hairy roots. Methods: Agrobacterium rhizogenes R15834, R1601, R1000, and A4 were used to infect V. segetalis leaves to induce hairy roots and the influences of different physicochemical factors on its growth were investigated. The content of vaccarin was determined by HPLC. Results: A. rhizogenes R1601 had the highest induction rate by converted into V. segetalis leaves, The best growth cycle of cell suspension culture was defined when cultured in liquid MS medium with pH value of 6 and sucrose concentration of 3%, vaccarin in V. segetalis hairy roots was slightly higher than that in the seed. Conclusion: V. segetalis could successfully induce hairy roots, the foundation has been established for further oplimizing the proper cultural system for V. segetalis hairy roots and regulating the secondary metabolites.
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OBJECTIVE:To conduct supplementary study for quality standard for Compound vaccariae tablets. METHODS:TLC was used for the qualitative identification of Vaccariae;HPLC was used to determine the contents of vaccarin and anthranilic acid. The column was YMC-Pack Pro C18 with mobile phase of methanol- 0.3% phosphpric acid(gradient elution)at the flow rate of 1.0 ml/min,the detection wavelength was 280 nm,column temperature was 35 ℃ and volume size was 10 μl. RESULTS:The chromatographic spots were clear and well-separated without interference from negative control. The linear range was 0.055 7-2.228 7μg(r=0.999 9)for vaccarin and 2.011 4-40.227 9 μg(r=0.999 9)for anthranilic acid;RSDs of precision,stability and reproduc-ibility tests were no more than 1.01%;average recoveries were respectively 99.69%(RSD=1.11%,n=6) and 99.84%(RSD=1.18%,n=6). CONCLUSIONS:The method is simple,rapid and accurate with good reproducibility,and can be used for quality control of Compound vaccariae tablets.