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@#Objective: To determine the inhibitory effects of pachymic acid on lung adenocarcinoma (LUAD) cells and elucidate its underlying mechanism. Methods: CCK-8, wound healing, Transwell, Western blot, tube formation, and immunofluorescence assays were carried out to measure the effects of various concentrations of pachymic acid on LUAD cell proliferation, metastasis, angiogenesis as well as autophagy. Subsequently, molecular docking technology was used to detect the potential targeted binding association between pachymic acid and protein tyrosine phosphatase 1B (PTP1B). Moreover, PTP1B was overexpressed in A549 cells to detect the specific mechanisms of pachymic acid. Results: Pachymic acid suppressed LUAD cell viability, metastasis as well as angiogenesis while inducing cell autophagy. It also targeted PTP1B and lowered PTP1B expression. However, PTP1B overexpression reversed the effects of pachymic acid on metastasis, angiogenesis, and autophagy as well as the expression of Wnt3a and β-catenin in LUAD cells. Conclusions: Pachymic acid inhibits metastasis and angiogenesis, and promotes autophagy in LUAD cells by modulating the Wnt/ β-catenin signaling pathway via targeting PTP1B.
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OBJECTIVE To investigate the effect of pachymic acid （PA） on renal function and fibrosis in chronic renal failure （CRF） rats and its potential mechanism based on the Ras homolog gene family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway. METHODS Using male SD rats as subjects， the CRF model was established by 5/6 nephrectomy； the successfully modeled rats were divided into model group， PA low-dose， medium-dose and high-dose groups （5， 10， 20 mg/kg PA）， high-dose PA+ROCK pathway activator lysophosphatidic acid （LPA） group （20 mg/kg PA+1 mg/kg LPA）， with 15 rats in each group. Another 15 rats were selected as the sham operation group with only the kidney exposed but not excised. The rats in each drug group were gavaged and/or injected with the corresponding liquid via the caudal vein， once a day， for 12 consecutive weeks. During the experiment， the general condition of rats was observed in each group. After the last administration， the serum renal function indexes （blood urea nitrogen， serum creatinine， uric acid） of rats in each group were detected， the renal histopathological changes were observed； the renal tubule injury score and the area of renal fibrosis were quantified. The levels of oxidative stress indexes [malondialdehyde （MDA）， superoxide dismutase （SOD）] and inflammatory factors （tumor necrosis factor-α， interleukin-1β， interleukin-6）， the positive expression rates of connective tissue growth factor （CTGF） and collagen Ⅰ were detected as well as the expression levels of pathway-related proteins （RhoA， ROCK1） and fibrosis- related proteins （transforming growth factor-β1， bare corneum homologs 2， α-smooth muscle actin） were determined. RESULTS Compared with the sham operation group， the rats in model group had reduced diet， smaller body size， listless spirit and sluggish response， reduced and atrophied glomeruli， dilated renal tubules with chaotic structure， and a large number of inflammatory cells infiltrated interstitium； the serum levels of renal function indexes， renal tubule injury score， renal fibrosis area proportion， the levels of MDA and inflammatory factors， the positive expression rates of CTGF and collagen Ⅰ， and the expression levels of pathway-related proteins and fibrosis-related proteins in renal tissues were significantly increased， while SOD level was significantly decreased （P＜0.05）. Compared with the model group， the general condition and pathological injuries of kidney tissue of rats in PA groups were improved to varying degrees，and the above quantitative indexes were significantly improved in a dose-dependent manner （P＜0.05）. LPA could significantly reverse the improvement effect of PA on the above indicators （P＜0.05）. CONCLUSIONS PA can improve renal function and alleviate renal fibrosis in CRF rats， which may be related to inhibiting the activation of RhoA/ROCK signaling pathway.
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Objective To explore the therapeutic effect and mechanism of pachymic acid(PA)on Helicobacter py-lori(Hp)-associated gastritis in rats.Methods A rat model of Hp-associated gastritis was established;all rats were separated into control group(CT group),model group(group M),PA low-dose group(PA L group),PA high-dose group(PA H group),and PA H+phosphatidylinositol 3-kinase(PI3K)activator(740 Y-P)group;the gastric mucosal injury index(UI)of rats in each group was evaluated,transmission electron microscopy was applied to observe the morphology of gastric mucosal cells.HE staining was applied to evaluate the pathological characteristics of gastric mucosa.ELISA was applied to detect the levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),IL-10,induc-ible nitric oxide synthase(iNOS),and superoxide dismutase(SOD)in gastric tissue.Western blot method was applied to detect the expression of PI3K,phosphorylated PI3K(p-PI3K),protein kinase B(AKT),p-AKT,nuclear factor(NF)-κB p65,and p-NF-κB p65 proteins.Results Compared with the CT group,the gastric mucosa erosion,epithelial ede-ma,congestion,and severe ulcers were observed in the group M,with epithelial cell pyknosis and inflammatory cell in-filtration,the UI,IL-6,TNF-α,iNOS,and the expression levels of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins increased,the levels of IL-10 and SOD decreased(P<0.05);compared with group M,the gastric mucosal damage and inflammatory cell infiltration in the PA L and PA H groups were improved,the UI,IL-6,TNF-α,iNOS by the host animal and the expression of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins all decreased,the level of IL-10 and SOD was increased(P<0.05);compared with the PA H group,the pathological damage of the gastric mucosa in the PA H+740 Y-P group was aggravated,with epithelial cell pyknosis.The UI,IL-6,TNF-α,iNOS,and the expression of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins increased,the levels of IL-10 and SOD decreased(P<0.05).Conclusions PA might facilitate the treatment of Hp-associated gastritis in rats by inhibiting the PI3K/AKT/NF-κB signaling pathway.
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OBJECTIVE@#To investigate the effect of pachymic acid (PA) against TNBS-induced Crohn's disease (CD)-like colitis in mice and explore the possible mechanism.@*METHODS@#Twenty-four C57BL/6J mice were randomized equally into control group, TNBS-induced colitis model group and PA treatment group. PA treatment was administered via intraperitoneal injection at the daily dose of 5 mg/kg for 7 days, and the mice in the control and model groups were treated with saline. After the treatments, the mice were euthanized for examination of the disease activity index (DAI) of colitis, body weight changes, colon length, intestinal inflammation, intestinal barrier function and apoptosis of intestinal epithelial cells, and the expressions of TNF-α, IL-6 and IL-1β in the colonic mucosa were detected using ELISA. The possible treatment targets of PA in CD were predicted by network pharmacology. String platform and Cytoscape 3.7.2 software were used to construct the protein-protein interaction (PPI) network. David database was used to analyze the GO function and KEGG pathway; The phosphorylation of PI3K/AKT in the colonic mucosal was detected with Western blotting.@*RESULTS@#PA significantly alleviated colitis in TNBS-treated mice as shown by improvements in the DAI, body weight loss, colon length, and histological inflammation score and lowered levels of TNF-α, IL-6 and IL-1β. PA treatment also significantly improved FITC-dextran permeability, serum I-FABP level and colonic transepithelial electrical resistance, and inhibited apoptosis of the intestinal epithelial cells in TNBS-treated mice. A total of 248 intersection targets were identified between PA and CD, and the core targets included EGFR, HRAS, SRC, MMP9, STAT3, AKT1, CASP3, ALB, HSP90AA1 and HIF1A. GO and KEGG analysis showed that PA negatively regulated apoptosis in close relation with PI3K/AKT signaling. Molecular docking showed that PA had a strong binding ability with AKT1, ALB, EGFR, HSP90AA1, SRC and STAT3. In TNBS-treated mice, PA significantly decreased p-PI3K and p-AKT expressions in the colonic mucosa.@*CONCLUSION@#PA ameliorates TNBS-induced intestinal barrier injury in mice by antagonizing apoptosis of intestinal epithelial cells possibly by inhibiting PI3K/AKT signaling.
Subject(s)
Animals , Mice , Mice, Inbred C57BL , Crohn Disease , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Interleukin-6 , Molecular Docking Simulation , Tumor Necrosis Factor-alpha , Colitis/chemically induced , Inflammation , Apoptosis , ErbB ReceptorsABSTRACT
ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.
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Objective To establish a new method and validate its feasibilities by the simultaneous quantitative assay of four triterpenes in Poria cocos. Methods A new quality evaluation method of multi-components by single marker (QAMS) was established and validated for P. cocos. Pachymic acid (PA), dehydropachymic acid (DPA), dehydrotumulosic acid (DTA), and dehydrotrametenolic acid (DMA) were selected as analytes while pachymic acid was chosen as internal reference substance to evaluate the quality. The relative correction factor (RCF) of pachymic acid to the other three triterpenes were calculated. The method was evaluated by the comparison of quantity between external standard method and QAMS method. Results The contents of four triterpenes in 17 batches of P. cocos from QAMS method were not significantly different from those from external standard method. Conclusion The method with a single marker is accurate and feasible to determine PA, DPA, DTA, and DMA when some authentic standard substance are unavailable.
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OBJECTIVE:To establish the method for the content determination of 4 active components in Compound xiaosuanzao chewable tablets.METHODS:HPLC-ELSD method was adopted.The determination was performed on a Grace Brava C18-BDS column with mobile phase consisted of acetonitrile-water (gradient elution) at the flow rate of 1.0 mL/min.The column temperature was 25 ℃,and sample size was 20 μL.The drift tube temperature is 100 ℃,and the carrier gas flow rate is 2.9 L/min.RESULTS:The linear ranges of betulic acid,betulinol,pachymic acid and glycyrrhizic acid were 44.50-890.0 μg/mL (r=0.999 3),20.28-405.6 μg/mL (r=0.999 7),20.50-656.0 μg/mL(r=0.999 7) and 10.50-336.0 μg/mL(r=0.999 6),respectively.RSDs of precision,stability and reproducibility were all lower than 3.0％.The recoveries were 99.44％-101.12％ (RSD=0.57％,n=6),99.41％-100.39％ (RSD=0.34％,n=6),99.31％-100.46％ (RSD=0.51％,n=6),98.96％-101.19％ (RSD=0.84％,n=6),respectively.CONCLUSIONS:The method is simple,precise,stable and reproducible,and can be used for simultaneous determination of 4 active components in Compound xiaosuanzao chewable tablets.
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Objective: To study the potential apoptotic effect of pachymic acid isolated from Poria cocos, a traditional Chinese medicinal fungi, on human breast cancer cells. Methods: Pachymic acid was isolated and purified from the crude ethanol extract of P. cocos. Human breast cancer cell line MDA-MB-231 cells were treated with pachymic acid. Relative cell viabilities were determined by the Cell Counting Kit-8 assay; Apoptosis was analyzed by Annexin V/PI dual staining with flow cytometry; The protein expression levels of poly ADP ribose polymerase (PARP) and other apoptotic related proteins were detected by Western blotting. Results: Pachymic acid reduced the viability of MDA-MB-231 cells dose-and time-dependently. Moreover, pachymic acid induced the apoptosis in MDA-MB-231 cells. The data of Western blotting showed that pachymic acid up-regulated the expression level of PARP, in a dose-dependent manner. In addition, apoptosis-related proteins like Bax, Bcl-2, and casapases were all affected by pachymic acid. Conclusion: Pachymic acid could induce the apoptosis in human breast cancer cells, which provides a novel information for clinical application of pachymic acid and its preparations for the breast cancer patients.
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Poria cocos is a well-known traditional Chinese traditional medicine (TCM) that grows around roots of pine trees in China, Korea, Japan, and North America. Poria cocos has been used in Asian countries to treat insomnia as either a single herb or part of an herbal formula. In a previous experiment, pachymic acid (PA), an active constituent of Poria cocos ethanol extract (PCE), increased pentobarbital-induced sleeping behaviors. The aim of this experiment was to evaluate whether or not PCE and PA modulate sleep architectures in rats as well as whether or not their effects are mediated through GABA(A)-ergic transmission. PCE and PA were orally administered to individual rats 7 days after surgical implantation of a transmitter, and sleep architectures were recorded by Telemetric Cortical encephalogram (EEG) upon oral administration of test drugs. PCE and PA increased total sleep time and non-rapid eye movement (NREM) sleep as well as reduced numbers of sleep/wake cycles recorded by EEG. Furthermore, PCE increased intracellular chloride levels, GAD65/67 protein levels, and alpha-, beta-, and gamma-subunits of GABA(A) receptors in primary cultured hypothalamic neuronal cells. These data suggest that PCE modulates sleep architectures via activation of GABA(A)-ergic systems. Further, as PA is an active component of PCE, they may have the same pharmacological effects.
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Animals , Humans , Rats , Administration, Oral , Asian People , China , Cocos , Electroencephalography , Ethanol , Eye Movements , Glutamate Decarboxylase , Japan , Korea , Medicine, Chinese Traditional , Neurons , North America , Pinus , Poria , Receptors, GABA-A , Sleep Initiation and Maintenance DisordersABSTRACT
Objective: To compare the content of chemical components in each drug in formula granule and traditional decoction of Rehmanniae Decoction with six ingredients. Methods: By using high performance liquid chromatography analysis and various chromatographic conditions, the contents were determined respectively, which were listed as follows: acteoside, loganin, paeonol, allantoin, pachymic acid and alisol B 23-acetate. Results: The contents of acteoside, loganin, paeonol, allantoin, pachymic acid and alisol B 23-acetate in traditional decoction were 304.5, 2 473.6, 3 135.1, 708.8, 5.9, and 104.4 μg/g, and they were 289.6, 3 685.7, 706.5, 714.2, 17.4, and 217.8 μg/g correspondingly in formula granule. The contents of acteoside and allantoin were basically the same between them; The contents of loganin, pachymic acid, and alisol B 23-acetate in formula granule were significantly higher than those in the traditional decoction; The content of paeonol in formula granule was significantly lower than that in the traditional decoction. Conclusion: The content difference of the chemical components is related to its chemical character between formula granule and traditional decoction.
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This study was investigated to know whether pachymic acid (PA), one of the predominant triterpenoids in Poria cocos (Hoelen) has the sedative-hypnotic effects, and underlying mechanisms are mediated via gamma-aminobutyric acid (GABA)-ergic systems. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also prolonged sleeping time, and reduced sleep latency showing synergic effects with muscimol (0.2 mg/kg) in shortening sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) doses. Additionally, PA elevated intracellular chloride levels in hypothalamic primary cultured neuronal cells of rats. Moreover, Western blotting quantitative results showed that PA increased the amount of protein level expression of GAD65/67 over a broader range of doses. PA increased alpha- and beta-subunits protein levels, but decreased gamma-subunit protein levels in GABA(A) receptors. The present experiment provides evidence for the hypnotic effects as PA enhanced pentobarbital-induced sleeping behaviors via GABA(A)-ergic mechanisms in rodents. Taken together, it is proposed that PA may be useful for the treatment of sleep disturbed subjects with insomnia.
Subject(s)
Animals , Mice , Rats , Administration, Oral , Blotting, Western , Cocos , gamma-Aminobutyric Acid , Hypnotics and Sedatives , Locomotion , Muscimol , Neurons , Pentobarbital , Poria , Receptors, GABA-A , Rodentia , Sleep Initiation and Maintenance DisordersABSTRACT
OBJECTIVE: To establish an analytical method to determine dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid and pachymic acid in poria quickly and accurately. METHODS: An UPLC method was established on an HSS T3 Column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of water (containing 0.05% phosphoric acid) and acetonitrile in gradient elution mode, and the detection wavelength was set at 210 nm. RESULTS: The standard curves of dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid, and pachymic acid showed good linearity in 5.400-108.0, 2.040-40.80, 5.020-100.4, 2.120-42.40, 5.060-101.2 and 5.100-102.0 μg · mL-1, respectively and the average recoveries were 98.0% with RSD of 2. 9% for dehydrotumulosic acid, 99. 0% with RSD of 2.8% for tumulosic acid, 101.5% with RSD of 2.5% for polyporenic acid C, 97.1% with RSD of 2.7% for 3-epi-dehydrotumulosic acid, 101.5% with RSD of 2.1% for dehydropachymic acid and 99.6% with RSD of 1.1% for pachymic acid, respectively. CONCLUSION: The method is quick, accurate, and can be used to determine multiple triterpenoid acids in Poria simultaneously.
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AIM: The contents of pachymic acid in Poria in different parts and in different localities were measured and compared. METHODS: 9 samples were measured with HPLC. RESULTS: The content of pachymic acid in Fushen is the highest in different parts and the content of pachymic acid in Guangxi province is the highest in five localities. CONCLUSION: The content of pachymic acid was obviously variable in different parts and this result made the basis of the application of with the different parts of Poria. The content of pachymic acid wasn't obviously variable in different localities.