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ObjectiveTo establish a neuroinflammation-based obesity and depression comorbidity （COM） model in mice and explore the pharmacodynamics and preliminary pharmacological mechanism of tripterine on COM mice. MethodC57BL/6J mice were randomly divided into a normal group （Chow）， a diet-induced obesity group （DIO）， and a COM group. The mice in the COM group were fed on a high-fat diet and chronically stressed with moist litter for 12 weeks to establish the COM model. C57BL/6J mice were randomly divided into a Chow group， a COM group， and a tumor necrosis factor-α（TNF-α） knock-down group. In the TNF-α knock-down group， TNF-α shRNA adeno-associated virus was injected into the amygdala through brain stereotaxis， and the expression of TNF-α in the amygdala was down-regulated. C57BL/6J mice were randomly divided into a Chow group， a DIO group， a DIO + low-dose tripterine group （0.5 mg·kg-1）， a DIO + high-dose tripterine group （1.0 mg·kg-1）， a COM group， a COM + low-dose tripterine group （0.5 mg·kg-1）， and a COM + high-dose tripterine group （1.0 mg·kg-1）. The body weight， food intake， glucose tolerance， white/brown fat ratio， serum total cholesterol （TC）， triglyceride （TG）， and high-/low-density lipoprotein cholesterol （HDL-C and LDL-C） content were recorded， and obesity of mice in each group was evaluated. Forced swimming test （FST）， tail suspension test （TST）， and open field test were used to evaluate the degree of depression of mice in each group. Immunofluorescence staining was used to detect the protein expression levels of neuropeptide Y， tryptophan hydroxylase 2 （TPH2）， and brain-derived neurotrophic factor （BDNF） in various brain nuclei of mice. Correlation analysis was used to detect the correlation of obesity and depression indexes. ResultThe comparison of the Chow group and the DIO group indicated that COM mice showed obesity and depression. To be specific， obesity was manifested as increased body weight and food intake （P<0.05， P<0.01）， as well as increased NPY expression in the central amygdala， and depression was manifested as prolonged immobility time in FST and TST （P<0.01）， and reduced TPH2-positive 5-hydroxytryptamine neurons in the dorsal raphe nucleus （DRN） and basolateral nucleus of the amygdala （BLA）. The down-regulation of TNF-α protein in BLA of COM mice shortened the immobility time in FST and TST （P<0.05， P<0.01）， increased TPH2/BDNF-positive neurons in BLA， and showed no significant changes in obesity. In DIO mice， the administration of 0.5 mg·kg-1 tripterine for 9 days significantly decreased the 60 min blood glucose in glucose tolerance （P<0.01） and food intake （P<0.05）. In COM mice， 1.0 mg·kg-1 tripterine was administered for 14 days to significantly decrease 30 min blood glucose in glucose tolerance （P<0.01）， and food intake （P<0.05）， and immobility time in TST （P<0.01）， increase TPH2-BDNF double-labeled cells in BLA and DRN， and reduce the area of TMEM119-stained cells. ConclusionThe model of obesity and depression comorbidity can be properly induced in mice under the condition of dual stress of energy environment. Tripterine can effectively interfere with obesity-depression comorbidity， and its mechanism may be related to the inhibition of central nervous system inflammation.
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The p21-activated kinase 1 (PAK1) is a member of the P21-activated protein kinase family that plays an important role in the proliferation and on cogenesis of pancreatic cancer. PAK1 is an important target for the treatment of pancreatic cancer. At present, akinase inhibitor targeting PAK1 is still in the preclinical research stage. Therefore, screening for new PAK1 kinase inhibitors is of great significance. In this study the natural compound celastrol was found to have a significant inhibitory effect on PAK1, with an IC50 value of 3.614 μmol·L-1. Molecular docking results showed that celastrol had good binding to PAK1. An MTT assay indicated that celastrol inhibited the proliferation of pancreatic cancer cells BxPC-3 and PANC-1. Mechanistic studies revealed that the inhibition of pancreatic cancer cells by celastrol was reversed by PAK1 siRNA. Celastrol inhibited PAK1 and the subsequent activation of downstream signaling pathways, thereby activating apoptosis signaling pathways and triggering apoptosis in pancreatic cancer cells. These findings suggested that celastrol induced apoptosis in pancreatic cancer cells by suppressing the PAK1 kinase signaling pathway and has potential value for the treatment of pancreatic cancer.
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Objective To improve the dissolution in vitro, thereby to prepare the solid dispersion (SD) from the extract of Tripterygium wilfordii (ETW). Methods Polyethylene glycol 6000 (PEG 6000) and poloxamer 188 (F68) were used as carrier to prepare ETW-SD by solvent-melting method. The triptolide, triptonide, wilforine, tripterine and wilforlide were used as the evaluation indexes to characterize the optimal prescription of ETW-SD by dissolution in vitro, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). Results The optimal formulation for ETW-SD composed of ETW-PEG 6000-F68 (1∶2∶1). Compared with the raw materials, the dissolution of triptonide, triptolide and wilforine increased by a factor of 3.32, and wilforine by 2 times, while the dissolution of tripterine and wilforlide reached more than 83% within 60 min.
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OBJECTIVE:To optimize the methanol extraction technology of tripterine in the fruit of Celastrus monospermus. METHODS:HPLC was adopted to determine the content of tripterine. Using extraction time,extraction times,solid-liquid ratio as investigation factors,extraction rate of tripterine as investigation index,orthogonal test was designed,and verification test was con-ducted. RESULTS:The optimal methanol extraction technology was as follow as 45% methanol extracting 2 h each time with sol-id-liquid ratio of 1:8 for 3 times. In the verification test,the average extraction rate of tripterine was 0.917 mg/g(RSD=2.85%, n=3). CONCLUSIONS:The optimized methanol extraction technology is stable and feasible with high extraction rate,and is suit-able for the extraction of tripterine in the fruit of C. monospermus.
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This study was aimed to prepare tripterine-coix seed component microemulsions (TCC-MEs) with coix seed oil and coix seed polysaccharide as bimodal excipients and evaluate its activity on anti-lung cancer in vivo.Coix seed oil was extracted by supercritical extraction methods and coix seed polysaccharide was obtained through hot water extraction and alcohol precipitation from coix seed.Then,coix seed oil was used as the oil phase,and coix seed polysaccharide aqueous solution was used as the aqueous phase.RH40 was used as the surfactant.PEG400 was used as cosurfactant to prepare microemulsion.TCC-MEs were made by water titration method and characterized by size,polymer dispersity index (PDI),zeta potential,encapsulation efficiency and drug loading capacity.And then,anti-tumor activity of TCC-MEs was evaluated on the xenograft Lewis tumor mouse models.The liver and kidney toxicity was evaluated by HE staining and biochemical indicators.The results showed that the optimized prescription for TCC-MEs was coix seed oil 400 mg,RH40 300 mg,PEG 400 100 mg,coix seed polysaccharide 50 mg,tripterine 10 mg;and the tripterine encapsulation efficiency was (90.72 ± 0.28)％;the drug loading capacity was (1.08 ± 0.17)％;the mean diameter of microemulsion was (43.86 ± 0.22) nm;PDI was 0.10 ± 0.01;the zeta potential was (-13.14 ± 1.35) mV.The activity of anti-lung cancer in TCC-MEs was significantly better than that of the control group,with no significant liver and kidney toxicity after treatment with TCC-MEs.It was concluded that the prepared TCC-MEs had advantages of small amount of conventional auxiliary materials,small particle size and high stability.This study showed that the combination of triptolide and coix seed to microemulsion system has synergistic and attenuated effect.
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To establish a LC-MS/MS method for determination of tripterine in Beagle plasma and study its pharmacokinetics after oral administration of tripterygium tablet. Plasma samples were extracted with dichloromethane and separated on a Phenomenex Luna C₈ (2.0 mm×50 mm, 3 μm) column with methanol-acetonitrile isopropanol(1∶1)-1‰formic acid (15∶55 ∶30) as the mobile phase. Tripterine ([M+H] ⁺, m/z 451.3/201.1) and internal standard prednisolone ([M+H] ⁺, m/z 361.1/147.1) were monitored in multiple reaction monitoring (MRM). The concentration-time curves were simulated by drug and statistic software 1.0 and the pharmacokinetic parameters were calculated. There was a good linear relationship between peak area ratio and concentration of tripterine and internal standard prednisolone within range of 0.680 0-136.0 μg•L⁻¹. The limit of quantitation was 0.680 0 μg•L⁻¹ and the intra- and inter-day precision was within 6.15%. The absolute recovery rate was between 50.42% to 51.65%. The concentration-time curves were consistent with the one-compartment model(w=1/cc). The main pharmacokinetic parameters after a single dose were as follows： Cmax (35.64±9.540) μg •L⁻¹,Tmax(2.62±0.69) h,T1/2(2.93±0.29) h, CL (0.308±0.056) L•kg⁻¹•h⁻¹, AUC0-12 (131.16±31.94) μg•L•h⁻¹, AUC0-∞ (142.83±37.57) μg•L•h⁻¹. The established LC-MS/MS method was proved to be sensitive, accurate and convenient, suitable for the pharmacokinetic study of Tripterygium tablet in Beagle dogs.
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Objective To clarify the inhibitory effect of Tripterine combined with Cisplatin on C6 glioma ceils and its apoptosis mechanism.Methods C6 glioma cells were treated with Tripterine,Cisplatin,or Tripterine combined with Cisplatin.CCK-8 assay was used to detect the growth inhibition rate in each group.Flow cytometry analysis was used to test the cell apoptosis rate.The expressions of apoptosis-related proteins including Bcl-2,Bax,XIAP,NF-kB were analyzed by ELISA.Results Compared with Tripterine-or Cisplatin-treated group,the inhibition ratio of cell growth in Tripterine and Cisplatin combination group was (69.76±7.28)％,which could significantly inhibit the growth of C6 glioma cells(t=23.78,P＜0.01).The apoptosis rate was significantly higher (47.75±5.63)％ in combination group than in Tripterine or Cisplatin treated group.The results of ELISA showed that the expression of Bax was significantly higher and Bcl-2,XIAP,NF-κB were obviously lower in the combination group than in Tripterine or Cisplatin treated group (t=35.27,P＜ 0.01).Conclusion The combination of Tripterine and Cisplatin significantly increases the inhibition rate on C6 glioma cells through upregulating Bax and inhibiting Bcl,XIAP and NF-kB to induce the apoptosis of C6 glioma cells.
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Objective To investigate the effect of tripterine on wear particle-induced inflammatory reaction by cell culture in vitro. Methods Wear particles from artificial joints were prepared by vacuum ball milling, and made into the par-ticle suspension by using serum-free medium. RAW264.7 cells were cultured, subcultured and divided randomly into four groups according to different treatment factors:blank control group (group A), wear particle group (group B), wear particle+tripterine group (group C) and tripterine group (group D). After 24 hours, the toxicity of tripterine was detected by CCK-8 as-say. ELISA was used to detect the expressions of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β). RT-PCR was used to detect the expressions of TNF-α, IL-1βand nuclear factor-κB (NF-κB) at the gene level. Western blot assay was used to detect the expression of NF-κB at the protein level. Results The results showed that 1 mg/L tripterine was little cytotoxic. The expressions of pro-inflammatory cytokines and the activation of NF-κB were significantly higher in wear particle group than those in blank control group (P＜0.05), which were significantly decreased after the treatment with tripter-ine (group C, P＜0.05). Conclusion Tripterine can inhibit the wear particle-induced expression and release of pro-inflam-matory cytokines at the gene level and protein level and the activation of NF-κB in RAW264.7 cells.
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Objective: To prepare the self-assembled beads drug delivery system of triptrine based on cyclodextrin and oil, and to carry out its in vitro evaluation. Methods: The beads were prepared by a continuously shaking starting from a mixture of cyclodextrin aqueous solution and oil. The bead diameter and drug distribution were investigated by microscopic observations, and drug disperse state was observed by differential scanning calorimetry (DSC). The drug-loading and entrapment efficiency of tripterine in beads were investigated by HPLC. The in vitro dissolution of tripterine in beads was also determined. Results: The diameter was in the range of (1.49 ± 0.20) mm. The drug-loading and encapsulation efficiency of the prepared beads were (87.21 ± 0.58) μg/g and (80.14 ± 1.24)%, respectively. DSC suggested that tripterine existed as amorphous form in beads. Confocal microcopy showed that the hydrophobic drug was localized inside the beads. The maximum cumulative release amount of tripterine in simulated intestinal fluid was more than 80% at 6 h. Conclusion: The formulation and preparation process are practical and simple, and these beads have great potentialities for carrying hydrophobic drug.
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OBJECTIVE: To prepare hydrophobic nano-CaCO3 by nano-CaCO3 and stearic acid. And to prepare tripterine solid dispersion using the new material as a carrier by solvent method. METHODS: The structure of modified nano-CaCO3 was characterized by TEM, DSC, XRD and FTIR. Meanwhile the solid dispersions were characterized by SEM, DSC, XRD and FTIR, and the in vitro dissolution test of tripterine was performed. RESULTS: When the ratio of drug to modified nano-CaCO3 was 1:4, tripterine could be dispersed amorphously in the carrier. And the accumulative drug-release percentage in vitro at 0.5 h was 7.05%. With the time increasing, the accumulative drug-release percentage also increased to 90.03% at 12 h. Moreover, tripterine solid dispersion had a better sustained release effect. CONCLUSION The nano-CaCO3 modified by stearic acid can be used as controlled-release carrier for tripterine.
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Objective To investigate the effect of tripterine on surface ultrastructure and proliferative activity of Burkitt lymphoma cell line Raji. Methods Raji cells were treated with different concentrations (0. 5,1. 0,1. 5,and 2. 0 μg/ml) of tripterine. Then the proliferation of Raji cells was determined by CCK8 assay, the cell membrane ultrastructure was analyzed by atomic force microscope, the apoptosis of cells was determined by Hoechst 33258 staining and flow cytometric analysis, and the cell cycle was assayed by flow cytometry. Results CCK8 assay showed that the survival rate of cells decreased from (80. 67 ± 2. 08)% to (38. 53 ± 2.25)% 24 h after treatment with different concentrations of tripterine. The cell survival rate decreased from (74. 17 ± 3. 20)% to (33. 22 ± 1. 64)% 48 h after treatment. Tripterine significantly inhibited the proliferation of Raji cells and the inhibition was in a time-and concentration-dependent manner. Atomic force microscope scan showed that the untreated Raji cells were round, with relatively smooth surface. After Raji cells were treated with 2. 0 μg/ml tripterine for 24 h and 48 h, the ultrastructure of the cell membrane was collapsed, and the cell surface was rough and uneven. Hoechst 33258 staining demonstrated apoptotic cells. Apoptotic rate of the Raji cells increased from (3. 50 ± 1. 73)% to (38. 27 ± 6. 05)% 24 h after treatment with different concentrations of tripterine. Flow cytometry analysis showed that 24 h after treatment with 1. 5 μg/ml tripterine, S phase Raji cells were significantly increased compared with control group (P<0. 05). Conclusion Our results demonstrate that tripterine can alter the cell membrane ultrastructure of Raji cells and can inhibit Raji cell proliferation through inducing cell apoptosis.
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Objective: To explore the molecular mechanism of HMC-1 cell apoptosis on exposure to tripterine. Methods: After the HMC-1 cells were incubated with tripterine, the expression of bcl-2, bax, bcl-X,c-myc and ICE were assayed by using immunohistochemical staining and RT-PCR. Results: The expression of bax,c-myc were up-regulated and bcl-2 down-regulated at protein level.The expression of bax,bcl-X L, especially bcl-2 were down-regulated, and ICE was up-regulated at mRNA level. Conclusion: These results suggest that apoptosis of HMC-1 cells induced by tripterine is regulated by different expression of apoptosis-related genes.
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Objective To study the protective effects of tripterine on experimental lupus nephritis glomerulosclerosis.Methods Different doses of tripterine were injected peritoneally to BW F1 mice at different stages.The levels of 24 hour urine protein excretion and serum anti dsDNA antibodies,and the expressions of renal collagen type Ⅰ,type Ⅳ,MMP 2,TIMP 2,and transforming growth factor (TGF) ? 1 mRNA were analyzed.Results ①Tripterine suppressed the development of proteinuria,decreased the level of serum anti dsDNA antibodies,reduced the local expressions of TGF ? 1,collagen type Ⅰ,type Ⅳ,TIMP 2 and improved the expression of MMP 2 in murine kidney.②The use of tripterine before occurence of proteinuria got more obvious protective effects than it did after the occurence of proteinuria.③No significance was found between both 3 mg/kg (a week) tripterine treated and 6 mg/kg (a week) groups.Conclusion Tripterine has a definite protective effect on glomerulosclerosis of the lupus murine model.The decrease of renal collagen type Ⅰ and type Ⅳ is probably due to its suppressive effects on the expression of local TGF ? 1,TIMP 2 and its improvement effect on the local expression of MMP 2.
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Objective To determine the content of tripterine in Tripterygium wilfordii and its tablets by HPLC.Methods An external standard method by HPLC with Zorbax C_(18)column as fixed phase and methanol-1% HAc(87∶13) as mobile phase was adopted.The detection wavelength was 425 nm and the flow rate was 1.0 mL/min.Results The linear range for tripterine was 40.96～204.8 ?g/mL(r=(0.999 6).) The average recovery of Chinese medicinal materials was 98.37% and RSD was 1.01%(n=9);the average recovery of preparation sample was 98.59% and RSD was 1.18%(n=9).Conclusion The method is simple and accurate,which can be adoptable for quantitative analysis of tripterine in the plants of Tripterygium L.