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Objective To explore the effect of shionone(SHI)on motor function in the mouse model of spinal cord injury(SCI)and probe into the underlying molecular mechanism.Methods C57BL/6 mice were treated to induce the SCI model and then assigned into a model group(SCI group),a SCI+SHI group,and a sham surgery(control)group.The Basso mouse scale(BMS)score was determined to evaluate the recovery of motor function in SCI mice.Hematoxylin-eosin(HE)staining,Nissl staining,and immunofluorescence staining were employed to examine the fibrosis,morphological changes of neurons,and neuron apoptosis in the spinal cord tissue of SCI mice,respectively.The mouse hippocampal neuronal cell line HT22 was cultured in vitro and then classified into tumor necrosis factor α(TNF-α)induction and SHI groups.Western blotting was employed to determine the expression of apoptosis-associated proteins.Network pharmacology,gene ontology annotation,and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were employed to predict the possible molecular targets and signaling pathways of SHI in promoting functional recovery from SCI.Furthermore,the prediction results were verified by in vitro and in vivo experiments.Results Compared with the SCI group,the SCI+SHI group showed increased BMS score on days 21,28,35,and 42(P=0.003,P=0.004,P=0.023,and P=0.007,respectively),reduced area of spinal cord fibrosis(P=0.021),increased neurons survived(P=0.001),and down-regulated expression of cleaved cysteine aspastic acid-specific protease 3(cleaved Caspase-3)(P=0.017).Compared with the TNF-α group,the SHI group presented down-regulated expression levels of cleaved Caspase-3 and Bax(P=0.010,P=0.001)and up-regulated expression level of Bcl-2(P=0.001).The results of bioinformatics analysis showed that SHI might improve the motor function of SCI mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.The results of in vivo and in vitro experiments showed that SHI inhibited the phosphorylation of PI3K and Akt in SCI mice or HT22 cells exposed to TNF-α(all P<0.05).The number of apoptotic HT22 cells after treatment with insulin-like growth factor 1 was higher than that in the SHI group(P=0.003).Conclusion SHI may inhibit neuron apoptosis via the PI3K/Akt signaling pathway,thereby promoting the recovery of motor function in SCI mice.
Subject(s)
Mice , Animals , Proto-Oncogene Proteins c-akt/metabolism , Caspase 3/metabolism , Phosphatidylinositol 3-Kinases , Tumor Necrosis Factor-alpha/metabolism , Mice, Inbred C57BL , Spinal Cord Injuries , Apoptosis , Neurons/pathology , FibrosisABSTRACT
Objective: To study the chemical constituents from the roots of Millettia speciosa (Leguminosae). Methods: Compounds in the 95% ethanol extract from the air-dried roots of M. speciosa were isolated by chromatography on silica gel column together with recrystallization, and their structures were identified by their physicochemical characteristics and spectral features. Results: Sixteen components were isolated from the air-dried roots of M. speciosa and identified as 7-oxo-β-sitosterol (1), aurantiamide acetate (2), shionone (3), maleic acid (4), psoralen (5), N-methylcytisine (6), lupeol caffeate (7), bisdemethoxycurcumin (8), vanillic acid (9), syringic acid (10), 6-methoxydihdyrosanguinarine (11), glycyrrhizic acid (12), (E)-3, 3'-dimethoxy-4, 4'-dihydroxystilbene (13), schisandrol B (14), 7-hydroxylathyrol (15), and nardosinone (16). Conclusion: All the compounds are isolated from this plant for the first time, and compounds 3, 7, 8, 11, and 13-16 are isolated from the plants of Leguminosae for the first time, compounds 2, 4-6, 9, 10 are isolated from the plants of Millettia Wight et Arn. for the first time.
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Objective: To optimize the extraction technology of Asteris Radix et Rhizoma in Zibai Zhisou Capsules. Methods: Orthogonal test was carried out. The influences of concentration of solvent, the dosage of solvent, duration of extraction, and frequency of extraction on extraction results were investigated by using the content of shionons as index. Results: The optimal extraction technique was to extract pueraria in 80% alcohol with 8 times the weight of herbal medicine for 3 times, with 60 min once. Conclusion: High yield of extractum and high content of shionon are obtained with the present technology. The results with better repeatability are stable, which can provide the reference for the production of Zibai Zhisou Capsules.
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Objective To develop a simple,sensitive,and accurate method for the determination of shionone in rat plasma after administration of shionone.Methods The separation was developed by HPLC on a Waters shieldTM RP18 column (150mm × 3.9mm,5 μm) with a mobile phase composed of acetonitrile-0.05％ phosphoric acid water (98:2) at a flow rate of 1.0 mL/min.UV Detector was set at 200nm.Results The linear range of the standard curves was 0.043-1.720 μg/ml with the correlation coefficient of 0.995.The intra-and inter-day precisions were all below 10％.Conclusion The developed method can be successfully applied to the pharmacokinetic study.
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Objective To develop a sensitive, simple, and accurate method for the determination of shionone in rat plasma after ig administration of Asteris Radix petroleum ether extract (RAPE). Methods The separation was achieved by HPLC on a RP18 column (150 mm × 3.9 mm, 5μm) with a mobile phase composed of acetotitrile-0.05% phosphoric acid water (98: 2) at a flow rate of 1.0 mL/min. UV Detector was set at 200 nm and friedelin was chosen as an internal standard. Results The linear range of the standard curves was (0.3443-22.0) μg/mL with the correlation coefficient of 0.9968. The intra- and inter-day precisions were all below 10% and the relative error was -3.5%-1.1%.Conclusion The developed method can be successfully applied to the pharmacokinetic study. After ig administration of RAPE, T1/2(ka) is (33.09 ± 7.32) min and T1/2(ke) is (84.95 ± 22.34) min.
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Objective To establish a method for determining the content of Shionone in Chantui Zhike Granules.Methods The determination of Shionone was performed at 490nm by TLC Scanning,with Petroleum ether∶ethyl acetate(19∶1)as developer and 10 %ethanol sulfate as color-developing agent.Results The linear range of Shionone was in the range of 0.96～5.76 ?g and r=0.9998.The average recovery was 98.67 %and RSD was 0.77 %.Conclusion This method is accurate with a good reproducibility and can be used for the quality control of Chantui Zhike Granules.
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Objective To establish a HPLC method for the determination of Shionone in Ziwan Zhisou granule. Methods Phenomenex C18 column (250? 4.6 mm) was used. The mobile phase was acetonitrile and the detection wave length was at 203 nm. Result A good linearity of Shionone was in the range of 0.025～ 0.250 ? g/? L, r=0.9996. The average recovery was 97.8 % , RSD was 2.37 % ( n=5) . Conclusion This method is sensitive, accurate and simple and with good reproducibility for the determination of Shionone in Ziwan Zhisou granule.