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MicroRNAs (miRNAs) crucially modulate fundamental biologic processes such as angiogenesis. In the present study, we focused on the molecular function of miRNA-370-3p (miR-370) in regulating the angiogenic activity of endothelial cells (ECs). Transfection with miR-370 mimic (miR-370m) significantly inhibited the sprouting of human dermal microvascular EC (HDMEC) and HUVEC spheroids and mouse aortic rings, whereas miR-370 inhibitor (miR-370i) promoted sprout formation. Additional in vitro assays demonstrated the pleiotropic inhibitory effects of miR-370m on HDMEC proliferation, migration, and tube formation. Moreover, Matrigel plugs containing miR-370m-transfected HDMECs exhibited a reduced microvessel density after implantation into CD1 nude mice when compared with controls. In contrast, miR-370i exerted proangiogenic effects. Mechanistic analyses revealed that miR-370 directly targets smoothened (SMO) and down-regulates bone morphogenetic protein (BMP)-2 expression in HDMECs. Accordingly, inhibition of SMO by cyclopamine reversed miR-370i-induced HDMEC proliferation and migration. In addition, BMP-2 treatment counteracted miR-370m-suppressed tube formation of HDMECs, whereas blockade of BMP-2 with neutralizing antibody significantly inhibited miR-370i-induced tube formation. Taken together, these novel findings indicate that miR-370 is a potent inhibitor of angiogenesis, which directly targets SMO and BMP-2.-Gu, Y., Becker, V., Zhao, Y., Menger, M. D., Laschke, M. W. miR-370 inhibits the angiogenic activity of endothelial cells by targeting smoothened (SMO) and bone morphogenetic protein (BMP)-2.

Objective Ischemic stroke is a major cause of death in the global population, with a high disability and mortality rate. Lack of regenerative ability is considered to be the fundamental cause. This study aims to determine the effect of Shh pathway, which mediates regenerative signaling in response to CNS injury, on myelin repair and Olig1 expression in focal ischemic lesions in the rat. Methods A model of middle cerebral artery occlusion (MCAO) was established using the intraluminal suture method where the middle cerebral artery (MCA) was restricted for 120 min. Cyclopamine, a specific inhibitor of Shh, or saline was administered 12 h after MCAO surgery and lasted for 7 days. After MCA occlusion, male Sprague-Dawley rats were randomly allocated to cyclopamine- or saline-treated groups. A group of no-injection animals after MCAO were used as controls. The Shh signaling pathway, myelinogenesis-related factor MBP and Olig1 were testedby immunohistochemistry and RT-PCR assay. Results The levels of Shh and its component Gli1 were elevated from 1 d up to 14 d following ischemia, indicating that the Shh-Gli1 axis was broadly reactivated. Treatment with cyclopamine can partially block the Shh signaling pathway, prevent myelin repair, and decrease the Olig1 expression following ischemic stroke. Conclusion That blockade of Shh signaling concurrently with the creation of a lesion aggravated ischemic myelin damage, probably via its downstream effects on Olig1 transcription. Shh plays a contributory role during regeneration in the CNS, thereby providing promising new therapeutic strategies to assist in recovery from ischemic stroke.

Objective This study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury. Methods Healthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation. Results The proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months’ intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant. Conclusion Hh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.

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.

Various studies have revealed that the Hedgehog (Hh) signaling pathway promotes ovarian cancer invasion, migration and drug resistance. Previous studies by our group have identified a set of genes, including multidrug resistance gene 1 (MDR1), that are regulated by Hh signaling in ovarian cancer. However, the association between Hh signaling activation and MDR1 expression requires further validation. In the present study, reverse transcription‑quantitative PCR or western blot assays were used to evaluate the mRNA and protein expression levels of MDR1, Sonic Hh (Shh), glioma‑associated oncogene 2 (Gli2), Gli1 and γ‑phosphorylated H2A.X variant histone (γ‑H2AX). MTT and colony‑formation assays were performed to determine the effect of cisplatin (DDP) after inhibiting the Hh pathway in ovarian cancer cells. The results indicated that MDR1, Gli2 and Shh levels were much higher in SK‑OV‑3 cells with acquired DDP resistance than in native SK‑OV‑3 cells. ES‑2 cells with overexpression of Gli2 were capable of efficiently forming colonies in the presence of low DDP concentrations. By contrast, Gli2 knockdown in SK‑OV‑3 cells decreased the colony‑forming ability under the same concentration of DDP. As determined by MTT assays, knockdown of Gli2 or targeting of the Hh signaling pathway with either Gli‑antagonist 61 (GANT61) or cyclopamine, in combination with DDP treatment, diminished the viability of ES‑2 and SK‑OV‑3 cells, whereas Gli2 overexpression increased the viability of ES‑2 cells in the presence of DDP. Knockdown of Gli2 or targeting the Hh signaling pathway with GANT61 also increased γ‑H2AX levels but decreased the expression of MDR1 in the presence of DDP. MDR1 expression is regulated by the Hh signaling pathway and is likely a downstream transcription factor of Gli2. In conclusion, targeting the Hh signaling pathway increases the sensitivity of ovarian cancer to DDP. MDR1 is a target gene of the Hh signaling pathway and this pathway may affect chemoresistance of ovarian cancer to DDP via MDR1.

Background Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear. Methods Realtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2. Results Specific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC. Conclusions TPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC. Graphical abstract

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Stromal‑epithelial interaction serves a pivotal role in normal prostate growth, as well as the onset of benign prostatic hyperplasia (BPH). The present study aimed to explore the role of cyclopamine in the proliferation and apoptosis of epithelial and stromal cells in rats with BPH by blocking the Hedgehog signaling pathway. Cyclopamine (an inhibitor of the Hedgehog signaling pathway) was administered in a rat model of BPH, and the expression of Ki67 (proliferation factor) was determined by immunohistochemistry. In addition, epithelial and stromal cells were separated and cultured in order to investigate the role of cyclopamine in the progression of BPH. The expression of Hedgehog signaling pathway‑ and apoptosis‑related genes, including basic fibroblastic growth factor (b‑FGF) and transforming growth factor β (TGF‑β), was evaluated using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Cell proliferation, cell cycle and apoptosis were analyzed using an MTT assay and flow cytometry. We identified upregulated Ki67 expression and activated Hedgehog signaling pathway in rats with BPH. Cyclopamine inhibited the activation of the Hedgehog signaling pathway. In response to cyclopamine treatment, epithelial and stromal cell proliferation was inhibited; this was concomitant with decreased Ki67, TGF‑β, and b‑FGF expression. On the other hand, epithelial cell apoptosis was enhanced, which was associated with increased Bax and reduced Bcl‑2 expression. Based on these findings, we proposed that cyclopamine may serve as a potential therapeutic agent in the treatment of BPH. Cyclopamine could inhibit epithelial and stromal cell proliferation, and induce epithelial cell apoptosis by suppressing the Hedgehog signaling pathway.

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9–mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.

The Sonic Hedgehog protein (Shh) has been extensively researched since its discovery in 1980. Its crucial role in early neurogenesis and endogenous stem cells of mature brains, as well as its recently described neuroprotective features, implicate further important effects on neuronal homeostasis. Here, we investigate its potential role in the survival, proliferation, and differentiation of neural precursors cells (NPCs) under inflammatory stress as a potential adjunct for NPC-transplantation strategies in spinal cord injury (SCI) treatment. To this end, we simulated an inflammatory environment in vitro using lipopolysaccharide (LPS) and induced the Shh-pathway using recombinant Shh or blocked it using Cyclopamine, a potent Smo inhibitor. We found that Shh mediates the proliferation and neuronal differentiation potential of NPCs in vitro, even in an inflammatory stress environment mimicking the subacute phase after SCI. At the same time, our results indicate that a reduction of the Shh-pathway activation by blockage with Cyclopamine is associated with reduced NPC-survival, reduced neuronal differentiation and increased astroglial differentiation. Shh might thus, play a role in endogenous NPC-mediated neuroregeneration or even be a potent conjunct to NPC-based therapies in the inflammatory environment after SCI.

Embryos and tumors share several characteristics, but embryos differ from tumors in their coordination of cellular- and tissue-level processes, including organized differentiation, remodeling of tissues through apoptosis, and disciplined migrations of cells. Embryonic cellular events are kept on track through orderly cell-cell communication via signal transduction pathways. If the pathways are disrupted, development is perturbed, and malformation may result. Despite profound differences between embryos and tumors, the study of one has benefited our understanding of the other. Using cyclopia as an example, the history of humans' beliefs concerning and reactions to this horrific malformation are explored. During the latter half of the 20th century, interest in cyclopic sheep from high pastures in western Idaho led to the discovery that cyclopia occurred after pregnant ewes foraged in fields containing corn lily (Veratrum californicum). Eventually, the proximate teratogen was identified as cyclopamine (a steroidal alkaloid). The teratogenic mechanism was identified as inhibition of the sonic hedgehog (Shh) signal transduction pathway. Alert cancer researchers noted that a prominent form of medulloblastoma (a devasting childhood brain tumor) overexpressed Shh. Cyclopamine effectively inhibited the tumor in mice and killed human medulloblastoma cells in vitro. Thus, over a 60-year period, a molecule causing hideous malformations and much emotional pain was discovered and then found capable of restraining a destructive tumor, potentially saving children's lives and sparing emotional devastation of their families. The success of identifying cyclopamine as a cause of cyclopia and a potential cure for medulloblastoma emerged from mechanistic research shared by multiple disciplines.

Side population (SP) cells harbor malignant phenotypes in cancer. The aim of this study was to identify genes that modulate the proportion of ovarian cancer SP cells. Using a shRNA library targeting 15,000 genes, a functional genomics screen was performed to identify genes whose suppression increased the SP percentage. The biological effects caused by alteration of those identified genes were investigated in vitro and in vivo. We found that suppression of MSL3, ZNF691, VPS45, ITGB3BP, TLE2, and ZNF498 increased the proportion of SP cells. Newly generated SP cells exhibit greater capacity for sphere formation, single cell clonogenicity, and in vivo tumorigenicity. On the contrary, overexpression of MSL3, VPS45, ITGB3BP, TLE2, and ZNF498 decreased the proportion of SP cells, sphere formation capacity and single cell clonogenicity. In ovarian cancer cases, low expression of MSL3, ZNF691 and VPS45 was related to poor prognosis. Suppression of these six genes enhanced activity of the hedgehog pathway. Cyclopamine, a hedgehog pathway inhibitor, significantly decreased the number of SP cells and their sphere forming ability. Our results provide new information regarding molecular mechanisms favoring SP cells and suggest that Hedgehog signaling may provide a viable target for ovarian cancer.

Epithelial‑mesenchymal transition (EMT) provides a valuable source of fibroblasts that produce extracellular matrix in airway walls. The Sonic hedgehog (SHH) signaling pathway plays an essential role in regulating tissue turnover and homeostasis. SHH is strikingly upregulated in the bronchial epithelia during asthma. Snail1 is a major target of SHH signaling, which regulates EMT and fibroblast motility. The present study was designed to ascertain whether the combination of house dust mite (HDM) and transforming growth factor β1 (TGF‑β1) could induce EMT via the SHH signaling pathway in human bronchial epithelial cells (HBECs). HBEC cultures were treated with HDM/TGF‑β1 for different periods of time. The involvement of SHH signaling and EMT biomarkers was evaluated by quantitative real‑time PCR, western blotting and immunofluorescence staining. Small‑interfering RNA (siRNA) for glioma‑associated antigen‑1 (Gli1) or cyclopamine was used to inhibit SHH signaling in HBECs. HBECs stimulated by HDM/TGF‑β1 exhibited morphological features of EMT. E‑cadherin (an epithelial marker) was decreased after a 72‑h exposure to HDM/TGF‑β1 compared to that in the control cells, and the expression of type I collagen and FSP1 (mesenchymal markers) was increased. HDM/TGF‑β1 activated the SHH signaling pathway in HBECs, which led to Gli1 nuclear translocation and the transcriptional activation of Snail1 expression. Moreover, gene silencing or the pharmacological inhibition of Gli1 ameliorated EMT. In summary, these findings suggest that HDM/TGF‑β1 may induce EMT in HBECs via an SHH signaling mechanism. Inhibition of SHH signaling may be a novel therapeutic method for preventing airway remodeling in asthma.

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality. AKI is characterized by tubular injury, inflammation, and vascular impairment. However, the role of interstitial fibroblasts in the pathogenesis of AKI is largely unknown. Here, we show that fibroblasts were activated, as defined by vimentin expression, at 1 h after AKI triggered by ischemia‐reperfusion injury (IRI). They rapidly entered the cell cycle with Ki‐67‐positive staining, which started at 1 h and peaked at 12 h after IRI, whereas tubular cell proliferation peaked at 3 d. The trigger for such an early activation of fibroblasts was identified as sonic hedgehog (Shh), which was rapidly induced in renal tubules and could target interstitial fibroblasts. Tubule‐specific knockout of Shh in mice inhibited fibroblast activation and aggravated kidney injury and functional decline after IRI. Likewise, pharmacologic inhibition of Shh signaling with cyclopamine also hindered fibroblast activation and exacerbated kidney damage. These studies uncover that tubule‐derived Shh triggers the early activation of fibroblasts, which is required for kidney repair and regeneration. Our findings for the first time illustrate a previously unrecognized importance of interstitial fibroblasts in conferring renal protection in AKI.—Zhou, D., Fu, H., Liu, S., Zhang L., Xiao, L., Bastacky, S. I., Liu, Y. Early activation of fibroblasts is required for kidney repair and regeneration after injury. FASEB J. 33, 12576–12587 (2019). www.fasebj.org

To investigate the role of sonic hedgehog (Shh) signaling and epithelial–mesenchymal transition (EMT) in bladder cancer progression and invasion. We cultured three bladder cancer cell lines, muscle-invasive T24 and 5637, and non-muscle-invasive KK47, in the presence of a recombinant-Shh (r-Shh) protein or cyclopamine, a Shh signaling inhibitor, to investigate proliferation and expression of EMT markers. Wound-healing assays and transwell assay were performed to evaluate cell invasion and migration. Mice were then inoculated with bladder cancer cells and treated with cyclopamine. Mouse tumor samples were stained for Shh signaling and EMT markers. R-Shh protein enhanced cell proliferation, whereas cyclopamine significantly suppressed cell proliferation, especially in invasive cancer (5637 and T24) (p

Background Liver fibrosis is a wound‐healing process of liver featured by the over‐deposition of extracellular matrix (ECM) and angiogenesis. However, the effective treatment is lacking. Procyanidin B2 (PB2) is a flavonoid extract abundant in grape seeds with anti‐oxidant, anti‐inflammatory and anti‐cancer properties. The present study aimed to determine effects of PB2 on liver fibrosis. Method The CCl4‐induced mouse liver fibrosis model and a human hepatic stellate cell (HSC) line (LX2 cells) were used to study the activation, ECM production and angiogenesis of HSCs through Western blotting analysis, immunohistochemistry, immunofluorescence staining, flow cytometry and tubulogenesis assay. A Hedgehog (Hh) pathway inhibitor (cyclopamine) and Smoothened agonist (SAG) were used to investigate the role of PB2 on Hh pathway. Results The results showed that PB2 could inhibit the proliferation and induce apoptosis of HSCs. PB2 could also down‐regulate the expressions of VEGF‐A, HIF‐1α, α‐SMA, Col‐1 and TGF‐β1 of HSCs in vivo and in vitro. The application of SAG and cyclopamine proved that PB2 targets on Hh pathway. Conclusions PB2 inhibited the Hh pathway to suppress the activation, ECM production and angiogenesis of HSCs, therefore reverses the progression of liver fibrosis in vivo and in vitro.

Purpose Bioactive glass–ceramic (BGC) coatings have been extensively studied and clinically used as bone substitute materials because of their osteogenesis, osteoinduction, and osteoconduction characteristics. Although the Hedgehog (Hh) signaling pathway plays an important role in skeletal development, the relationship between BGC coatings and the Hh signaling pathway is unknown. Methods In this study, a BGC coating is fabricated by furnace sintering, and its surface is investigated by a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Furthermore, the expression of Ki67 is evaluated using immunofluorescence, and osteogenesis-related factors and Hh signaling pathway molecules on the BGC coating are examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blotting in bone marrow mesenchymal stem cells (BMSCs). Results The SEM and the TEM show that the BGC coating surface is smooth, without cracks, and composed of particles with mesoporous structure. The expression of Ki67 positive BMSCs of the BGC group is higher than that of the control group. Real-time RT-PCR and Western blotting assay reveal that the expression levels of osteoblast-related genes (BMP2, Osteocalcin, ALP, Runx2) and Hh signaling pathway molecules (Gli1, Smo) are much higher for the BGC coating group than those for the control group. Furthermore, after treating with Smo inhibitor cyclopamine, the Smo and Gli1 expressions in BMSCs are dramatically down-regulation for the BGC coating compared to those for the control group. Both mRNA and protein expression levels of osteogenesis-related factors was downregulated after treating Smo inhibitor cyclopamine in BMSCs with the BGC coating. Conclusions The BGC coatings promote osteogenesis probably via the Hh signaling pathway, which provides a theory reference for future clinical application of bone formation.

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.

BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. MATERIAL AND METHODS Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. RESULTS Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P

Background Previous research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods Hepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor β 1 (TGFβ1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results We found that hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFβ1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

Liver kinase B1 (LKB1) is revealed to have a close relationship with cancers. However, the underlying mechanism remains unclear. This study aimed to reveal the mechanism through which LKB1 performed its role in lung cancer. LKB1 shRNA and overexpression plasmid were employed to investigate the role of LKB1 in lung cancer in vitro and in vivo. The involvement of Shh signaling pathway was assessed by western blot. Silence of LKB1 promoted, while overexpression of LKB1 inhibited the migration, invasion and angiogenesis of lung cancer cells. Further study showed that Shh signaling pathway was suppressed by LKB1. Cyclopamine, a Shh signaling pathway inhibitor, reduced the effects of LKB1 silence, indicating that LKB1 inhibits the migration, invasion and angiogenesis through suppressing the Shh signaling pathway. In vivo study also showed that Shh signaling pathway was involved in the modulation of LKB1 on the metastasis and angiogenesis of lung cancer. Our study demonstrates that LKB1 inhibits the metastasis and angiogenesis of lung cancer through suppressing the Shh signaling pathway.

Procyanidin B2 (PB2), a naturally occurring flavonoid abundant in a wide range of fruits, has been shown to exert antioxidant, anti‐inflammatory and anticancer properties. However, the role of PB2 in the prevention of cold stimulation (CS)‐induced liver injury. The present study was undertaken to determine the effects of PB2 on liver injury induced by cold stimulation and its potential molecular mechanisms. The present study results showed that treatment with PB2 significantly reduced CS‐induced liver injury by alleviating histopathological changes and serum levels of alanine transaminase and aspartate transaminase. Moreover, treatment with PB2 inhibited secretion of inflammatory cytokines and oxidative stress in cold‐stimulated mice. PB2 reduced cold stimulation‐induced inflammation by inhibiting TLR4/NF‐κB and Txnip/NLRP3 signalling. Treatment with PB2 reduced oxidative stress by activating Nrf‐2/Keap1, AMPK/GSK3β signalling pathways and autophagy. Furthermore, simultaneous application of Shh pathway inhibitor cyclopamine proved that PB2 targets the Hh pathway. More importantly, co‐treatment with PB2 and cyclopamine showed better efficacy than monotherapy. In conclusion, our findings provide new evidence that PB2 has protective potential against CS‐induced liver injury, which might be closely linked to the inhibition of Shh signalling pathway.

The sonic hedgehog (Shh) pathway is highly activated in a variety of malignancies and plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. Our recent study showed that the inhibitors of the Shh pathway such as cyclopamine (CP), a Smothened (SMO) inhibitor, and GANT61, a Gli1 inhibitor, have modest inhibitory effects on thyroid tumor cell proliferation and tumor growth. The objective of this study was to determine whether autophagy was induced by inhibition of the Shh pathway and could negatively regulate GANT61-induced apoptosis. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 induced autophagy in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines; whereas Gli1 overexpression suppressed autophagy. Mechanistic investigation revealed that inhibition of the Shh pathway activated TAK1 and its two downstream kinases, the c-Jun-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). GANT61-induced autophagy was blocked by TAK1 siRNA and the inhibitors of TAK1 (5Z-7-oxozeaenol, 5Z), JNK (SP600125), and AMPK (Compound C, CC). Inhibition of autophagy by chloroquine and 5Z and by TAK1 and Beclin-1 siRNA enhanced GANT61-induced apoptosis and its antiproliferative activity. Our study has shown that inhibition of the Shh pathway induces autophagy by activating TAK1, whereas autophagy in turn suppresses GANT61-induced apoptosis. We have uncovered a previously unrecognized role of TAK1 in Shh pathway inhibition-induced autophagy and apoptosis.

Multi-infarct dementia (MID), a prominent subtype of vascular dementia (VD), is responsible for at least 15 to 20 percent of dementia in the elderly. Mitochondrial dysfunctions and glutamate neurotoxicity due to chronic hypoperfusion and oxidative stress were regarded as the major risk factors in the pathogenesis. Kaixin San (KXS), a classic prescription of Beiji Qianjin Yaofang, was applied to treatment for “amnesia” and has been demonstrated to alleviate the cognitive deficit in a variety of dementias, including MID. However, little is known whether mitochondria and glutamate are associated with the protection of KXS in MID treatment. The aim of this study was to investigate the role of KXS in improving the cognitive function of MID rats through strengthening mitochondrial functions and antagonizing glutamate neurotoxicity via the Shh/Ptch1 signaling pathway. Our data showed that KXS significantly ameliorated memory impairment and hippocampal neuron damage in MID rats. Moreover, KXS improved hippocampal mitochondrial functions by reducing the degree of mitochondrial swelling, increasing the mitochondrial membrane potential (MMP), and elevating the energy charge (EC) and ATP content in MID rats. As expected, the concentration of glutamate and the expression of p-NMDAR1 were significantly reduced by KXS in the brain tissue of MID rats. Furthermore, our results showed that KXS noticeably activated the Shh/Ptch1 signaling pathway which was demonstrated by remarkable elevations of Ptch1, Smo, and Gli1 protein levels in the brain tissue of MID rats. Intriguingly, the inhibition of the Shh signaling pathway with cyclopamine significantly inhibited the protective effects of KXS on glutamate-induced neurotoxicity in PC12 cells. To sum up, these findings suggested that KXS protected MID rats from memory loss by rescuing mitochondrial functions as well as against glutamate neurotoxicity through activating Shh/Ptch1 signaling pathway.

Ongoing chronic fibrosis and hypertrophy of the ligamentum flavum (LF) is an important cause of lumbar spinal canal stenosis (LSCS). Our previous work showed that WNT1-inducible signaling pathway protein 1 (WISP-1) is a critical driver of LF fibrosis. However, the potential mechanism has not been explored. Here, we found that Gli1 was upregulated in hypertrophic LF tissues and required for fibrogenesis in fibroblasts. Moreover, mechanical stretching increased the expression of WISP-1 in LF fibroblasts. Furthermore, WISP-1 induced fibrogenesis in vitro through the Hedgehog-Gli1 pathway. This conclusion was supported by the fact that WISP-1 activated the Hedgehog-Gli1 pathway in LF fibroblasts and that cyclopamine attenuated the effect of WISP-1-induced fibrogenesis. WISP-1 also promoted the transition of fibroblasts into myofibroblasts via the Hedgehog pathway. Importantly, a hypertrophic LF rabbit model induced by mechanical stress also showed pathological changes in fibrosis and elevated expression of WISP-1, Gli1, and α-SMA. Therapeutic administration of cyclopamine reduced collagen expression, fibroblast proliferation, and myofibroblast differentiation and ameliorated fibrosis in the mechanical stress-induced rabbit model. Collectively, our findings show mechanical stress/WISP-1/Hedgehog signaling as a new fibrotic axis contributing to LF hypertrophy and identify Hedgehog signaling as a therapeutic target for the prevention and treatment of LF fibrosis., Back pain: stopping signaling could soothe sore spine A signaling pathway that triggers fibrosis and abnormal growth (hypertrophy) of ligaments in the spine could be targeted to treat an age-related lumbar condition. In lumbar spinal canal stenosis, the ligamenta flava (LF), literally the ‘yellow ligaments’ between vertebrae, expand due to hypertrophy and fibrosis. This narrows the canal carrying the spinal cord, leaving sufferers in significant pain. Previously, Xinhui Liu and co-workers at the Affiliated Jiangning Hospital with Nanjing Medical University, China, demonstrated that the WISP-1 protein drives LF fibrosis and is highly expressed in hypertrophic LF. Now, the team have shown that expression levels of WISP-1 increase with mechnical stretching of LF cells over time. WISP-1 then activates a signaling pathway that promotes LF cell proliferation and the differentiation of fibroblast cells, inducing fibrosis. Inhibiting this signaling pathway could partially prevent the progression of stress-induced LF fibrosis.

OBJECTIVE: To explore the role of silent information regulator 1 (SIRT1)-mediated Sonic Hedgehog (SHH) pathway in reduced uterine perfusion pressure (RUPP) model of preeclampsia (PE) in rats. METHODS: The pregnant rats were divided into sham, RUPP, RUPP + rSIRT1 (recombinant SIRT1 protein), RUPP + rSHH (recombinant SHH protein), and RUPP + rSIRT1+ Cy (cyclopamine, an SHH pathway inhibitor) groups, followed by the determination of mean arterial pressure (MAP) and pregnancy outcome. The gene or protein expression was determined by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), or Western blotting. RESULTS: RUPP rats showed increases MAP with the lower levels of vascular endothelial growth factor (VEGF) and nitrite and nitrate (NOx), as well as the higher levels of soluble FMS-like tyrosine kinase-1 (sFlt-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in maternal plasma, which was attenuated after rSIRT1 or rSHH treatment. Besides, the improvement in the pregnancy outcome was seen in the rats from the RUPP + rSIRT1/rSHH groups as compared with the RUPP group. However, the therapeutic effect of rSIRT1 was reversed by cyclopamine. Placenta tissues of RUPP rats manifested the down-regulations of SIRT1, Patched-1 (PTCH1), and GLI family zinc finger 2 (GLI2), which were up-regulated in the RUPP + rSIRT1 group. CONCLUSION: SIRT1 was down-regulated while SHH pathway was inhibited in the placental tissue of PE rats. SIRT1 improved the blood pressure, angiogenic imbalance, inflammation, and pregnancy outcome in PE rats via SHH pathway, supporting its potential use for the treatment of PE.

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity,ift57, ift88, andift172mutants, which lack cilia, were crossed with the double-transgenic zebrafish strainTg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.

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 in vivo 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) (P < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist (P < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor (P < 0.05). In vivo 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 (P < 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.

Aim: Pathologic hyperplasia of fibroblast is responsible for the progression of intraarticular fibrosis. Laminin α4 (LAMA4), a subunit of laminin macromolecule family, was found to be overexpressed in various fibrotic tissues. However, the role of LAMA4 in knee arthrofibrosis remains elusive. Therefore, the aim of this study was to investigate the effect and mechanism of LAMA4 on fibroblast proliferation and migration. Materials and methods: Following knee surgery, LAMA4 expression was detected in intraarticular fibrous tissues in rabbits at week 2 and week 4, respectively. In lentivirus-mediated LAMA4-overexpressed fibroblasts, cellular proliferation was assessed by EdU labeling and cell cycle analysis, cellular migration was evaluated using Transwell assay, and the expressions of key components in Shh/Gli1 signaling were detected by qRT-PCR, western blot and immunofluorescence analysis. Additionally, canonical Shh cascade was further blocked in LAMA4-overexpressed fibroblasts by cyclopamine, and the changes in cellular proliferation and migration were investigated. Results: LAMA4 expression was positively correlated with the severity of knee arthrofibrosis. Functional studies demonstrated that LAMA4 overexpression facilitated proliferation, cell cycle progression and migration in fibroblasts. Mechanically, LAMA4 activated the canonical Shh/Gli1 signaling and promoted the nuclear translocation of Gli1 to upregulate expression of genes associated with cellular proliferation and migration. Intriguingly, blockage of Shh/Gli1 signaling with cyclopamine reversed the promoting effects of LAMA4 on proliferation and migration of fibroblasts. Conclusions: LAMA4 positively regulated cellular proliferation and migration in fibroblasts via activating the Shh/Gli1 signaling. LAMA4/Shh/Gli1 signaling axis might be a potential therapeutic target for the prevention of surgery-induced intraarticular fibrosis.