OBJECTIVE: To investigate the effect of piperlongumine(PL) on the proliferation and ferroptosis of human adriamycin-resistant chronic myeloid leukemia K562/ADR cells, and to explore its possible molecular mechanism. METHODS: CCK-8 assay was used to detect the effect of PL on the survival rate of K562/ADR cells and to screen the appropriate drug concentration. After K562/ADR cells were treated with low, medium and high concentrations of PL(2, 4, and 6 μmol/L), EdU proliferation assay and plate colony formation assay were used to detect cell proliferation and colony formation ability. CCK-8 assay was used to detect the effects of different inhibitors (Fer-1, Z-VAD, Nec-1) combined with PL on cell proliferation. The intracellular Fe²⁺, ROS, malondialdehyde(MDA) and glutathine(GSH) contents were respectively detected by iron ion colorimetry, DCFH-DA fluorescent probe, MDA and GSH kits. RT-qPCR and Western blot were respectively used to detect the expression level of GPX4 mRNA and protein in cells. Bioinformatics websites predicted miRNA that could target and regulate GPX4 . RT-qPCR was used to detect the effects of different concentrations of PL on the expression levels of the predicted miRNA. Dual luciferase gene reporter assay was used to verify the targeting relationship between miR-214-3p and GPX4 . After treating cells with PL or PL+miR-214-3p inhibitor, the Fe²⁺, ROS, MDA, GSH centents and GPX4 protein expression levels in cells were detected. RESULTS: PL inhibited K562/ADR cell proliferation in a concentration-dependent manner（r =0.979）. Compared with the blank control group, the survival rate, EdU positive cells rate in low, medium and high concentration PL groups were significantly decreased (P < 0.01). Compared with the PL group alone, the survival rate of cells in the Z-VAD+PL group was increased slightly (P < 0.05). The cell survival rate was significantly increased in medium or high concentration PL+Fer-1 group (P < 0.01). Compared with blank control group, ROS expression level in low concentration PL group was slightly increased (P < 0.05), and GSH content was slightly decreased (P < 0.05). In medium and high concentration PL groups, the contents of Fe²⁺, ROS and MDA were significantly increased (P < 0.01), while the contents of GSH, expression of GPX4 mRNA and protein were significantly decreased(P < 0.01). Bioinformatics prediction and double luciferase reporter gene experiment confirmed the targeting relationship between GPX4 and miR-214-3p. Compared with the blank control group, the expression level of miR-214-3p in cells of medium and high concentration PL groups was significantly increased (P < 0.01). Compared with PL group alone, the intracellular Fe²⁺, ROS and MDA contents in PL+miR-214-3p inhibitor group were all decreased (P < 0.01), while GSH content and GPX4 protein expression levels were significantly increased (P < 0.01). CONCLUSION Medium and high concentrations of PL can inhibit the proliferation of K562/ADR cells by inducing ferroptosis, which is related to the regulation of miR-214-3p pathway.
Humans ; Ferroptosis/drug effects* ; MicroRNAs/metabolism* ; Dioxolanes/pharmacology* ; Cell Proliferation/drug effects* ; K562 Cells ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Reactive Oxygen Species ; Doxorubicin/pharmacology* ; Signal Transduction ; Piperidones

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by congenital bilateral malformation of the large toe and progressive, extensive, and irreversible heterotopic ossification (HO) of soft tissues throughout the body, leading to severe disabilities. FOP is caused primarily by mutations in activin A receptor type 1 (ACVR1), also known as activin-like kinase 2 (ALK2), which encodes a receptor belonging to the bone morphogenetic protein (BMP) type I family. However, the continuous and complex process of HO in FOP is not yet fully understood, which has impeded the development of therapeutic drugs. Despite surgical removal of HO, which often results in recurrence and expansion of ossification, there is currently no definitive drug treatment available to completely prevent, halt, or reverse the progression of HO in FOP. Currently, researchers are intensively studying the pathogenesis of FOP at various stages and developing promising drug candidates, including saracatinib, palovarotene, and rapamycin. This review provides an overview of progress in understanding the mechanism of FOP and the development of therapeutic drugs, with the goal of providing insights for further research and the development of new treatment methods.
Myositis Ossificans/genetics* ; Humans ; Activin Receptors, Type I/genetics* ; Ossification, Heterotopic ; Mutation ; Sirolimus/therapeutic use* ; Quinolones/therapeutic use* ; Benzodioxoles/therapeutic use* ; Animals ; Quinazolines/therapeutic use*

A liquid chromatography-tandem mass spectrometry method was established and validated for determining the concentrations of costunolide(CO), piperine(PI), agarotetrol(AG), glycyrrhizic acid(GL), vanillic acid(VA), and glycyrrhetinic acid(GA) in rat plasma. This method was then applied to the toxicokinetic study of these six compounds in rats with chronic cerebral ischemia(CCI) following multiple oral doses of Zhachong Shisanwei Pills. Finally, the effects of continuous multiple-dose administration of Zhachong Shisanwei Pills on the liver of CCI rats were investigated. The results showed that after oral administration of different doses of Zhachong Shisanwei Pills, the in vivo exposure of AG, VA, and GA was relatively high, with AUC_(0-∞) values ranging from 604.0-2 494.2, 1 305.4-4 634.5, and 2 177.5-4 045.7 h·ng·mL~(-1), respectively, while the exposure of CO, PI, and GL was relatively low, with AUC_(0-∞) values ranging from 37.8-238.2, 2.4-17.0, and 146.9-408.5 h·ng·mL~(-1), respectively. The C_(max) and AUC_(0-∞) of the six compounds were positively correlated with the administered dose. The T_(max) of PI and AG ranged from 0.3 to 2.0 h, their T_(1/2) ranged from 0.8 to 2.9 h, and their mean residence time(MRT) ranged from 1.0 to 3.7 h. The T_(max) of GL and VA was shorter(0.4-1.9 h), while their T_(1/2)(2.6-5.9 h) and MRT(2.5-8.5 h) were longer. Both CO and GA exhibited a bimodal phenomenon, with T_(max) ranging from 1.6 to 6.6 h, T_(1/2) ranging from 2.8 to 7.7 h, and MRT ranging from 4.1 to 12.9 h. Liver histopathology after 28 days of continuous multiple-dose administration of Zhachong Shisanwei Pills showed that the liver tissue remained normal at a low dose(crude drug 0.8 g·kg~(-1), approximately 5 times the clinical equivalent dose). However, as the dose increased(crude drug 1.1-3.0 g·kg~(-1), 6.9-18.8 times the clinical equivalent dose), varying degrees of liver damage were observed. Blood biochemical tests revealed no significant changes in the serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), and total bile acid(TBA) in CCI rats from administration groups 1 to 3(crude drug 0.8, 1.1, 1.5 g·kg~(-1)). However, ALT, AST, ALP, and TBA levels in groups 4 and 5(crude drug 2.1, 3.0 g·kg~(-1)) showed significant increases. This study preliminarily elucidated the toxicokinetic characteristics of the six compounds in Zhachong Shisanwei Pills and their effects on liver tissue in CCI rats, providing data as a reference for clinical use.
Animals ; Tandem Mass Spectrometry/methods* ; Rats ; Drugs, Chinese Herbal/toxicity* ; Male ; Rats, Sprague-Dawley ; Brain Ischemia/blood* ; Chromatography, Liquid/methods* ; Polyunsaturated Alkamides/blood* ; Piperidines/toxicity* ; Benzodioxoles/toxicity* ; Alkaloids/blood* ; Liquid Chromatography-Mass Spectrometry

This article is to investigate the effect of piperine on the small intestine of mice with Parkinson's disease with dementia(PDD). Ninety-six C57 BL/6 mice of SPF grade were randomly divided into 8 groups(male, 12 in each group): normal group, model group, autophagy inhibitor group(6-amino-3-methylpurine, 3 MA, 30 mg·kg~(-1)), autophagy activator group(rapamycin, 1 mg·kg~(-1)), low, medium, and high dose piperine groups(10, 20, 40 mg·kg~(-1)), and medopar group(112.5 mg·kg~(-1)). Except for the normal group, mice in each group were injected subcutaneously with reserpine(0.1 mg·kg~(-1)) once every 48 hours for 40 days. In addition, on the 20 th day of administration, except for the normal group, the mice in the other groups were subjected to bilateral common carotid artery occlusion to finally prepare PDD models. At the same time, each group was given the corresponding drug treatment once a day for 40 days. After the last administration, the behavioral changes of mice were observed by autonomic activity experiment and hot plate experiment. The expression levels of α-synuclein(α-syn) and tyrosine hydroxylase(TH) in the small intestine were detected by immunohistochemistry. The expression levels of beclin-1, microtubule-associated protein 1 light chain 3 B(LC3 B) and p62 in the small intestine were detected by immunofluorescence assay. Hematoxylin-eosin staining was used to observe the pathological morphology of small intestine tissues in each group. Enzyme-linked immunosorbent assay was adopted for detection of β-amyloid precursor protein(APP), p-tau, acetylcholine transferase(ChAT), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in small intestine. Real-time fluorescent quantitative polymerase chain reaction was used to detect the expression of α-syn, TH, beclin-1, microtubule-associated protein 1 light chain 3(LC3), and p62 mRNA and mmu-miR-99 a-5 p in the small intestine. The results of this study showed that, as compared with the model group, the number of activities, the expression levels of ChAT, TH, and p62 were significantly increased in the 3 MA group, the various piperine dose groups, and the medopar group(P<0.05), and their first foot licking time was shortened; APP, p-tau, IL-6, TNF-α, α-syn, beclin-1, LC3 B and mmu-miR-99 a-5 p expression levels were significantly reduced(P<0.05). However, as compared with the model group, the number of activities, ChAT, TH, and p62 expression levels in the rapamycin group were significantly reduced(P<0.05), and the APP, p-tau, IL-6, TNF-α, α-syn, beclin-1, LC3 B and mmu-miR-99 a-5 p expression levels were significantly increased(P<0.05). As compared with the 3 MA group, the number of activities, ChAT, TH, and p62 expression levels were significantly reduced in the low and medium dose piperine groups and rapamycin group(P<0.05); howe-ver, their first foot licking time was significantly prolonged, APP, p-tau, IL-6, TNF-α, α-syn, beclin-1, LC3 B and mmu-miR-99 a-5 p expression levels were increased significantly(P<0.05). As compared with the medopar group, the number of activities, ChAT, TH, and p62 expression levels were significantly reduced in low dose piperine group and rapamycin group(P<0.05), but their first foot licking time was significantly extended, and APP, p-tau, IL-6, TNF-α, α-syn, beclin-1, LC3 B and mmu-miR-99 a-5 p expression levels were significantly increased(P<0.05). In addition, as compared with the normal group, the small intestinal epithelial cells of the model group and the rapamycin group were shed off a lot, with severe damages of intestinal mucosa as well as edema and shedding of the small intestine villi. After administration of the therapeutic interventions, the small intestinal epithelial cells of the 3 MA group, each dose group of piperine, and the medopa group were slightly damaged and the villi were slightly shed off. In summary, piperine has a protective effect on the small intestine of PDD model mice, showing reduced expression of mmu-miR-99 a-5 p, pro-inflammatory factors and autophagy factors, and the mechanism of slowing PDD pathological symptoms may be related to the inhibition of autophagy.
Alkaloids ; Animals ; Autophagy ; Benzodioxoles ; Dementia ; Intestine, Small ; Male ; Mice ; Parkinson Disease ; Piperidines ; Polyunsaturated Alkamides

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.
Alkaloids ; chemistry ; pharmacology ; Animals ; Benzodioxoles ; chemistry ; pharmacology ; Benzyl Compounds ; chemistry ; pharmacology ; Cholecalciferol ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Humans ; Kaempferols ; chemistry ; pharmacology ; Melanins ; genetics ; metabolism ; Monophenol Monooxygenase ; genetics ; metabolism ; Pigmentation ; drug effects ; Piperidines ; chemistry ; pharmacology ; Polyunsaturated Alkamides ; chemistry ; pharmacology ; Purines ; chemistry ; pharmacology ; Scopoletin ; chemistry ; pharmacology ; Vitiligo ; drug therapy ; enzymology ; metabolism ; Zebrafish

Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides in Panax ginseng. However, the oral bioavailability of Rh2 is low, with P-glycoprotein (P-gp) and CYP3A4 being reported to be the main factors. The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2. The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system, respectively. The pharmacokinetics of oral Rh2 (10 mg·kg) administered alone or in combination with piperine (10 and 20 mg·kg) was performed in rats. The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine. The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2. The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose (20 mg·kg) when compared to the control group, with relative bioavailability of 196.8%. The increase of Rh2 exposure led to increased serum levels of IL-12. In conclusion, piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.
Administration, Oral ; Alkaloids ; administration & dosage ; Animals ; Benzodioxoles ; administration & dosage ; Biological Availability ; Caco-2 Cells ; Cytochrome P-450 CYP3A ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Ginsenosides ; administration & dosage ; pharmacokinetics ; Humans ; Interleukin-2 ; metabolism ; Panax ; chemistry ; Piperidines ; administration & dosage ; Polyunsaturated Alkamides ; administration & dosage ; Rats ; Rats, Sprague-Dawley

BACKGROUNDBronchiolitis obliterans syndrome (BOS) often develops in transplant patients and results in injury to the respiratory and terminal airway epithelium. Owing to its rising incidence, the pathogenesis of BOS is currently an area of intensive research. Studies have shown that injury to the respiratory epithelium results in dysregulation of epithelial repair. Airway epithelial regeneration is supported by stromal cells, including fibroblasts. This study aimed to investigate whether the supportive role of lung fibroblasts is altered in BOS.

METHODSSuspensions of lung cells were prepared by enzyme digestion. Lung progenitor cells (LPCs) were separated by fluorescence-activated cell sorting. Lung fibroblasts from patients with BOS or healthy controls were mixed with sorted mouse LPCs to compare the colony-forming efficiency of LPCs by counting the number of colonies with a diameter of ≥50 μm in each culture. Statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., USA). The paired Student's t-test was used to test for statistical significance.

RESULTSLPCs were isolated with the surface phenotype of CD31-CD34-CD45- EpCAM+Sca-1+. The colony-forming efficiency of LPCs was significantly reduced when co-cultured with fibroblasts isolated from patients with BOS. The addition of SB431542 increased the colony-forming efficiency of LPCs to 1.8%; however, it was still significantly less than that in co-culture with healthy control fibroblasts (P < 0.05).

CONCLUSIONThe epithelial-supportive capacity of fibroblasts is impaired in the development of BOS and suggest that inefficient repair of airway epithelium could contribute to persistent airway inflammation in BOS.

Animals ; Benzamides ; pharmacology ; Bronchiolitis Obliterans ; metabolism ; pathology ; Cells, Cultured ; Coculture Techniques ; Dioxoles ; pharmacology ; Fibroblasts ; cytology ; drug effects ; metabolism ; physiology ; Flow Cytometry ; Humans ; Mice ; Stem Cells ; cytology ; drug effects ; metabolism

In 2015, fourteen topics were selected as major research advances in gynecologic oncology. For ovarian cancer, high-level evidence for annual screening with multimodal strategy which could reduce ovarian cancer deaths was reported. The best preventive strategies with current status of evidence level were also summarized. Final report of chemotherapy or upfront surgery (CHORUS) trial of neoadjuvant chemotherapy in advanced stage ovarian cancer and individualized therapy based on gene characteristics followed. There was no sign of abating in great interest in immunotherapy as well as targeted therapies in various gynecologic cancers. The fifth Ovarian Cancer Consensus Conference which was held in November 7–9 in Tokyo was briefly introduced. For cervical cancer, update of human papillomavirus vaccines regarding two-dose regimen, 9-valent vaccine, and therapeutic vaccine was reviewed. For corpus cancer, the safety concern of power morcellation in presumed fibroids was explored again with regard to age and prevalence of corpus malignancy. Hormone therapy and endometrial cancer risk, trabectedin as an option for leiomyosarcoma, endometrial cancer and Lynch syndrome, and the radiation therapy guidelines were also discussed. In addition, adjuvant therapy in vulvar cancer and the updated of targeted therapy in gynecologic cancer were addressed. For breast cancer, palbociclib in hormone-receptor-positive advanced disease, oncotype DX Recurrence Score in low-risk patients, regional nodal irradiation to internal mammary, supraclavicular, and axillary lymph nodes, and cavity shave margins were summarized as the last topics covered in this review.
Biomedical Research/*trends ; Breast Neoplasms/therapy ; Combined Modality Therapy ; Dioxoles ; Endometrial Neoplasms/therapy ; Female ; Genital Neoplasms, Female/genetics/*therapy ; Humans ; Immunotherapy ; Neoadjuvant Therapy ; Neoplasm Recurrence, Local ; Ovarian Neoplasms/prevention & control/therapy ; Papillomavirus Vaccines ; Precision Medicine ; Tetrahydroisoquinolines ; Uterine Cervical Neoplasms/prevention & control/therapy/virology ; Uterine Neoplasms/therapy

A series of [1,3]dioxolo[4,5-f]isoindolone derivatives were designed, synthesized and evaluated as inhibitors of acetylcholinesterases (AChE). Furthermore, their effects on memory impairment of mice induced by scopolamine were investigated with step-through test. The results suggested that most of the target compounds exhibited potential inhibition on AChE with IC50 values at micromolar range. Compounds I1 (IC50 value of 0.086 μmol · L(-1)) and I2 (IC50 value of 0.080 μmol · L(-1)) showed the strongest AChE inhibitory activity, which are equipotent to donepezil (IC50 value of 0.094 μmol · L(-1)). Moreover, compounds I1-I4 could improve the memory impairment induced by scopolamine in mice.
Animals ; Cholinesterase Inhibitors ; chemical synthesis ; chemistry ; Dioxoles ; chemical synthesis ; chemistry ; Drug Design ; Indans ; Inhibitory Concentration 50 ; Isoindoles ; chemical synthesis ; chemistry ; Memory Disorders ; drug therapy ; Mice ; Piperidines ; Scopolamine Hydrobromide