-
Gene Sep 2020This study aims to investigate the roles of Sonic hedgehog (Shh) signaling pathway in the occurrence and progression of Myelodysplastic Syndrome (MDS) and further...
OBJECTIVE
This study aims to investigate the roles of Sonic hedgehog (Shh) signaling pathway in the occurrence and progression of Myelodysplastic Syndrome (MDS) and further evaluate using jervine as therapeutic strategy for MDS by inhibiting Shh pathway.
METHODS
CD34+ cells from the bone marrow of 53 MDS patients were counted by flow cytometry and isolated by magnetic bead sorting. Shh, Smo, Ptch-1 and Gli-1 (involved in Shh pathway) in CD34+ cells were examined by RT-qPCR. Besides, the relationship between Shh pathway-related genes and the clinical features or prognosis of MDS were analyzed. Further, the effects of jervine on MUTZ-1 cells regarding their proliferation, apoptosis and cell cycle as well as Shh pathway-related gene and protein expression were analyzed.
RESULTS
Gene expression level of Shh, Gli-1 and Smo was significantly increased in MDS patients. Herein, Smo and Gli-1 were correlated with chromosome karyotype classification and IPSS. MDS patients with high expression of Smo or Gli-1 had a poor prognosis. Jervine inhibited gene and protein expression of Shh, Smo, Ptch-1 and Gli-1. Besides, jervine suppressed the proliferation and promoted the apoptosis of MUTZ-1 cells, as well as inhibited the transition of cells from G1 to S phase.
CONCLUSION
Shh signaling pathway of MDS patients is abnormally activated and participated in the occurrence and progression of MDS. Jervine intervention is a potential therapeutic strategy for MDS.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Male; Middle Aged; Myelodysplastic Syndromes; Patched-1 Receptor; Prognosis; Signal Transduction; Smoothened Receptor; Tumor Cells, Cultured; Veratrum Alkaloids; Young Adult; Zinc Finger Protein GLI1
PubMed: 32526259
DOI: 10.1016/j.gene.2020.144881 -
Frontiers in Immunology 2020Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs...
Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) ( < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist ( < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor ( < 0.05). study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling ( < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.
Topics: Arthritis, Rheumatoid; Biomarkers; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Female; Flow Cytometry; Hedgehog Proteins; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Male; Matrix Metalloproteinase 1; Middle Aged; Synoviocytes; Veratrum Alkaloids
PubMed: 32670287
DOI: 10.3389/fimmu.2020.01300 -
Xenobiotica; the Fate of Foreign... Oct 2020Peimine is a major component of widely used herb in pediatric. It is very common in Chinese traditional medicine to combine with two or more herbs in the clinic. To...
Peimine is a major component of widely used herb in pediatric. It is very common in Chinese traditional medicine to combine with two or more herbs in the clinic. To investigate the effect of peimine on the activity of cytochrome P450 enzymes (CYP450) is necessary for the clinical application of peimine.The effects of peimine on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in human liver microsomes (HLMs) with the specific inhibitors as positive control and without peimine or inhibitors as negative control. The enzyme kinetic parameters were calculated.It was found that peimine inhibited the activity of CYP3A4, 2E1, and 2D6 in a concentration-dependent manner with the values of 13.43, 21.93, and 22.46 μM, respectively. The inhibition of CYP3A4 was performed in a non-competitive manner with the value of 6.49 μM, and the inhibition of CYP2E1 and 2D6 was performed in a competitive manner with values of 10.76 and 11.95 μM. Additionally, peimine inhibited the activity of CYP3A4 in a time-dependent manner with the value of 6.17/0.049 minμM.Peimine inhibited the activity of CYP3A4, 2E1, and 2D6, which indicated the potential interaction between peimine and drugs metabolized by CYP3A4, 2E1, and 2D6. Further studies are needed to verify the drug-drug interaction and the effects.
Topics: Cevanes; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Liver; Microsomes, Liver
PubMed: 32338127
DOI: 10.1080/00498254.2020.1761572 -
Bioscience Reports May 2020Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was...
Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3'-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.
Topics: Adult Stem Cells; Animals; Apoptosis; Cartilage, Articular; Cell Proliferation; Cells, Cultured; Chondrocytes; Hedgehog Proteins; MicroRNAs; Multigene Family; Parathyroid Hormone-Related Protein; Patched-1 Receptor; Primary Cell Culture; Rabbits; Veratrum Alkaloids
PubMed: 32319512
DOI: 10.1042/BSR20191654 -
Bioscience Reports Oct 2022Peimine, a bioactive substance isolated from Chinese medicine Fritillaria, can potentially suppress pulmonary fibrosis (PF); however, its therapeutic mechanism remains...
Peimine, a bioactive substance isolated from Chinese medicine Fritillaria, can potentially suppress pulmonary fibrosis (PF); however, its therapeutic mechanism remains unclear. Recent evidence suggests the participation of M2-type macrophages in the pathogenesis of PF. The present study aimed to investigate the effect of peimine on a bleomycin (BLM)-induced PF rat model and the underlying mechanism of this effect. After BLM administration, peimine was administered to rats from day 29 to day 42, with pirfenidone (PFD) as a positive control. H&E and Masson's trichrome stain were used to analyze histological changes. Q-PCR and western blotting were used to measure mRNA levels and protein levels, respectively. High-throughput RNA sequencing (RNA-seq) technology detected the differentially expressed genes (DEGs) regulated by peimine. Our results revealed that peimine treatment significantly ameliorated BLM-induced PF by suppressing histological changes and collagen deposition. In addition, peimine decreased the number of M2 macrophages and the expression of profibrotic factors. RNA-seq results showed that DEGs regulated by peimine in IL-4-induced macrophages were mainly associated with immune system processes, the PI3K/Akt pathway, and the MAPKs pathway. Then, immunofluorescence assay and western blot results demonstrated that peimine treatment suppressed the expression of p-p38 MAPK and p-Akt (s473) and also inhibited the nuclear translocation of p-STAT6. In conclusion, the present study demonstrated that peimine has a protective effect on PF through the suppression of M2 polarization of macrophages by inhibiting the STAT6, p38 MAPK, and Akt signals.
Topics: Animals; Bleomycin; Cevanes; Collagen; Interleukin-4; Macrophages; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; RNA, Messenger; Rats; STAT6 Transcription Factor; p38 Mitogen-Activated Protein Kinases
PubMed: 36111628
DOI: 10.1042/BSR20220986 -
Curcumin- and Cyclopamine-Loaded Liposomes to Enhance Therapeutic Efficacy Against Hepatic Fibrosis.Drug Design, Development and Therapy 2020Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix...
BACKGROUND AND PURPOSE
Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix (ECM). Inhibition of HSC activation may be an effective treatment. Since various pathways control HSC activation, a combination of drugs with different mechanisms may be more effective than monotherapy.
METHODS
Here, we prepared liposomes loaded with curcumin and cyclopamine to inhibit HSC activation. We systematically analyzed the physicochemical characteristics of liposomes loaded with the two drugs, as well as their effects on HSC proliferation, activation and collagen production on gene, protein and cellular levels.
RESULTS
The prepared liposomes helped solubilize both drugs, contributing to their uptake by cells. Liposomes loaded with both drugs inhibited cell proliferation, migration and invasion, as well as induced more apoptosis and perturbed the cell cycle more than the free combination of both drugs in solution or liposomes loaded with either drug alone. Liposomes loaded with both drugs strongly suppressed HSC activation and collagen secretion.
CONCLUSION
Our results suggest that liposome encapsulation can increase the uptake of curcumin and cyclopamine as well as the synergism between them in anti-fibrosis. This approach shows potential for treating hepatic fibrosis.
Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Collagen; Curcumin; Liposomes; Liver Cirrhosis; Rats; Veratrum Alkaloids
PubMed: 33380787
DOI: 10.2147/DDDT.S287442 -
Pharmaceutical Biology Dec 2021Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
CONTEXT
Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
OBJECTIVE
To investigate the protective effect and mechanism of EDT in ulcerative colitis (UC).
MATERIALS AND METHODS
UC model was induced by 3% dextran sulphate sodium (DSS) solution for 7 days, meanwhile, EDT and Smoothened (Smo) inhibitor cyclopamine (Cyc) were intraperitoneally injected. In the first experiment, C57BL/6 mice divided into blank control, DSS, DSS + EDT (20 or 40 mg/kg) groups. In second experiment, added Cyc (5 mg/kg) and EDT + Cyc groups. All mice were sacrificed on day 8. Disease activity index (DAI), colon length and colon histology as well as MDA levels, SOD, and GSH-Px activities were measured. The expression of Sonic hedgehog (Shh), Patched, Smo, glioblastoma-1, zonula occludens-1 (ZO-1), occludin, cleaved caspase 3, Bax and Bcl-2 in colon was detected using RT-PCR and Western blotting.
RESULTS
After EDT treatment, compared with the DSS group, DAI (2.33 ± 0.516 vs. 3.67 ± 0.516), colon shortening (5.27 ± 0.476 vs. 4.53 ± 0.528 cm) and histological score (6.67 ± 1.211 vs. 12 ± 1.265) was significantly decreased. EDT also reduced inflammation, oxidative stress and apoptosis in colon. Additionally, EDT increased the expression of the tight junction proteins ZO-1 (35%) and occludin (66.3%). Mechanistically, EDT upregulated the Shh signalling pathway. However, Cyc-mediated inhibition of the Shh pathway partially abolished the effects of EDT.
DISCUSSION AND CONCLUSIONS
These results indicate EDT attenuates DSS-induced colitis by activating the Shh pathway. Further clinical trials are needed to demonstrate its efficacy on UC.
Topics: Animals; Colitis, Ulcerative; Colon; Cytokines; Dextran Sulfate; Flavanones; Hedgehog Proteins; Inflammation; Male; Mice; Mice, Inbred C57BL; Models, Animal; Oxidative Stress; Signal Transduction; Veratrum Alkaloids
PubMed: 34348563
DOI: 10.1080/13880209.2021.1948066 -
Cell Death & Disease Mar 2021Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor...
Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor outcomes on the current therapeutic regimes using Imatinib and its variants. It has been derived so far that leukemic stem cells (LSCs) are responsible for Imatinib resistance and CML progression. Sonic hedgehog (Shh) signaling has been implicated in proliferation of this Imatinib-resistant CD34(+) LSCs. Our work here identifies the molecular mechanism of Shh-mediated mutation-independent Imatinib resistance that is most relevant for treating CML-variants and non-compliant patients. Our results elucidate that while Shh can impart stemness, it also upregulates expression of anti-apoptotic protein-Bcl2. It is the upregulation of Bcl2 that is involved in conferring Imatinib resistance to the CD34(+) LSCs. Sub-toxic doses of Bcl2 inhibitor or Shh inhibitor (<
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Chromosomes, Human; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Up-Regulation; Veratrum Alkaloids
PubMed: 33707419
DOI: 10.1038/s41419-021-03542-w -
Life Sciences Sep 2020Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates...
AIMS
Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates in the development of various tissues and organs. However, the effect of Pax9 on atherosclerosis and VSMCs and the underlying mechanisms remain unclear.
MAIN METHODS
Western blotting was performed to assess Pax9 expression in atherosclerosis and VSMCs. Pax9 siRNA and overexpression plasmid were constructed to explore the biological function. Cell proliferation assay, phalloidin staining, and Transwell assay, accompanied by the sonic hedgehog (Shh) signaling pathway antagonist, cyclopamine (5 μM) and agonist, SAG (100 nM), were used to evaluate the VSMC phenotype, proliferation, and migration, as well as explore the associated mechanisms.
KEY FINDINGS
We first discovered Pax9 to be significantly increased in atherosclerotic mice and platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Pax9 knockdown inhibited the phenotypic transformation, proliferation, and migration of VSMCs, whereas the opposite effect was observed when Pax9 was overexpressed. Next, we established that Shh was activated in PDGF-BB-induced VSMCs. Moreover, Pax9 overexpression further activated Shh and exacerbated the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. These changes were effectively inhibited by treatment with the Shh signaling pathway antagonist. Consistently, Pax9 knockdown down-regulated Shh expression and inhibited the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. Treatment with the Shh signaling pathway agonist prevented these changes.
SIGNIFICANCE
Pax9 regulated VSMC phenotypic transformation, proliferation, and migration via Shh, which may represent a novel target for the treatment of atherosclerosis.
Topics: Animals; Atherosclerosis; Becaplermin; Cell Movement; Cell Proliferation; Cyclohexylamines; Gene Knockdown Techniques; Hedgehog Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; PAX9 Transcription Factor; Phenotype; Signal Transduction; Thiophenes; Veratrum Alkaloids
PubMed: 32634424
DOI: 10.1016/j.lfs.2020.118053 -
Gene Sep 2019Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and...
Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.
Topics: Alkaloids; Contig Mapping; DNA, Complementary; Gene Expression Regulation, Plant; Gene Library; Gene Ontology; Genetic Markers; High-Throughput Nucleotide Sequencing; Microsatellite Repeats; Molecular Sequence Annotation; Oligonucleotide Array Sequence Analysis; Plant Leaves; Plant Proteins; Plant Roots; Sequence Analysis, RNA; Steroids; Transcriptome; Veratrum
PubMed: 31288057
DOI: 10.1016/j.gene.2019.143962