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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.

Background The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood. Methods We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well‐known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK‐8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT‐PCR and Western Blot. Results We found up‐regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low‐dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells. Conclusions Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer., In this manuscript, we show that SHH signaling was active in ovarian cancer tissues. Inhibition of SHH signaling by cyclopamine (Cyp) enhanced the autophagy and promoted autophagic flux in ovarian cancer cell lines through PI3K/AKT signaling pathway. Our findings provided the experimental basis for simultaneously inhibiting the SHH pathway and autophagy as a new effective therapeutic strategy for patients with ovarian cancer.

Purpose: To investigate the effect of the hedgehog (Hh) pathway inhibitor, cyclopamine, and activator purmorphamine on articular cartilage cell proliferation. Methods: Rats were subjected to AA and CIA models. Secondary paw swelling was measured at 12, 15, 18, 21, 24, 27, and 30 days. The rats were sacrificed on day 30. Tissues from the cartilage and knee joints were collected. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay while cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide assay. Protein expression levels of Shh, Ptch1 and Gli1 were determined by Western blotting. Results: Compared with the control group, arthritis index and secondary foot swelling of the adjuvant arthritis (AA) and collagen-induced arthritis (CIA) groups deteriorated significantly (p < 0.05). MTT data revealed that cyclopamine promoted articular cartilage cell proliferation of the AA and CIA groups. The cell proliferation rates of AA and CIA groups were significantly higher than that of control group (p < 0.05). Flow cytometry showed that the cell apoptosis rates of AA and CIA groups were significantly lower than that of control group (p < 0.05). Compared with control, cyclopamine decreased the protein expression levels of sonic Hh, patched homologue 1 and glioma-associated oncogene homologue, but the effect of purmorphamine was the reverse. Conclusion: Hh pathway inhibitor (cyclopamine) and activator (purmorphamine) affect the expression of Hh pathway. Disruption of the Hh pathway may be of potential therapeutic significance in protecting articular cartilage from rheumatoid arthritis.

Endometrial carcinoma is a frequently occurring malignancy of the female reproductive tract. Previous investigations have implicated the Hedgehog signaling pathway, including the smoothened (Smo) receptor, in tumor progression. Here, we established a nude mouse xenograft model of endometrial cancer to investigate the effect of the Smo receptor antagonist cyclopamine on the growth of endometrial carcinoma. Mice were randomly sorted into two groups receiving 0.2 mL/mouse every other day of either cyclopamine (20 mg/mL) or solvent (control). The growth of the mice was observed for 18 days. Then, mice were euthanized, tumors were removed and weighed, and tumor volume inhibition rate (VIR) and weight inhibition rate (WIR) were calculated. Smo, Gli1, and Gli2 mRNA expression was measured in tumor tissues by RT-PCR, and Vascular endothelial growth factor (VEGF) and CD31 were detected by immunohistochemistry. We found that cyclopamine treatment reduced the tumor growth rate and significantly reduced the volumes and weights of the transplanted tumors compared with those of the controls. Furthermore, the transplanted tumors of cyclopamine-treated mice possessed lower expression levels of Smo, Gli1, and Gli2, and displayed a lower positive expression of VEGF and CD31. Thus, the Smo receptor antagonist cyclopamine inhibited transplanted tumor growth in nude mice with endometrial carcinoma, which is likely connected with cyclopamine downregulation of Smo, Gli1, and Gli2 mRNA expression that promote angiogenesis.

TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis.

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.

Wen Cao,1 Cong Zhang,1 Rong Chen,2,3 Qianqian Wu,1 Renhao Xu,2,3 Lan Zhang,1 Xiangjian Zhang1– 3 1Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, People’s Republic of China; 2Hebei Collaborative Innovation Center for Cardio-Cerebrovascular Disease, Shijiazhuang, Hebei 050000, People’s Republic of China; 3Hebei Vascular Homeostasis Key Laboratory for Neurology, Shijiazhuang, Hebei 050000, People’s Republic of ChinaCorrespondence: Xiangjian ZhangDepartment of Neurology, Second Hospital of Hebei Medical University, 215 Hepingxi Road, Shijiazhuang, Hebei 050000, People’s Republic of ChinaTel +86 15803210578Fax +86 31166002822Email zhang666xj@sina.comPurpose: Strokes are devastating as there are no current therapies to prevent long-term neurological deficits. Previous studies reported that cerebroprotein hydrolysate (CH) plays a role in neuronal protection in acute phase after ischemic stroke, while the long-term effects of CH upon brain plasticity and neurological outcomes after stroke are still uncertain. To address these gaps, we assessed the effect of a new cerebroprotein hydrolysate, CH1, on long-term gray and white matter integrity as well as axonal plasticity in the late phase after ischemic stroke and the potential mechanisms.Methods: Adult male mice were subjected to permanent distal middle cerebral artery occlusion (dMCAO), followed by daily intraperitoneal injection of CH1 for 14 days. Motor function was measured weekly through behavioral neurological evaluations. Gray matter intensity and white matter intensity were examined by immunofluorescence staining. The sonic hedgehog (Shh) inhibitor cyclopamine (CYC) was injected to determine the involvement of the Shh pathway in the therapeutic effects of CH1.Results: We found that intraperitoneal delivery of CH1, compared to vehicle administration, significantly improved long-term neurological outcomes at various times and promoted neuronal viability at 14 days but not at 28 days after stroke. Importantly, CH1 mitigated stroke-induced white matter injury and facilitated axonal plasticity in the late stage after stroke.Conclusion: These results unveil a previously unappreciated role for CH in the repair of white matter and brain plasticity after stroke.Keywords: ischemic stroke, cerebroprotein hydrolysate, axonal plasticity, white matter integrity, Shh pathway

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.

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.

BackgroundChondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO) model of OA.MethodsOA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1β as an inflammatory insult.ResultsTamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. Hypertrophic – IHH – and inflammatory – COX-2 – markers co-localized in OA cartilage samples.ConclusionsTamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

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

Context Placental dysfunction is the underlying cause of common major disorders of pregnancy, such as fetal growth restriction and preeclampsia. However, the mechanisms of placental dysfunction are not entirely elucidated. We previously reported 10 reliable preeclampsia pathways based on multiple microarray data sets, among which was the sonic hedgehog (SHH) pathway. In this study, we describe the significant role of SHH signaling involved in placental development and fetal growth. Design The placental expression levels of surrogate markers of the SHH pathway, patched homolog 1 (PTCH1) and glioma-associated oncogene homolog (GLI) 2, were evaluated using quantitative real-time PCR, western blot analysis, and immunohistochemistry. We investigated the underlying mechanisms of the SHH pathway in trophoblast syncytialization, a critical process for placental development and maturation, using primary cytotrophoblasts. Moreover, the potential roles of placental SHH signaling in the regulation of the IGF axis were explored by pathway analysis of microarray data. Finally, the influence of SHH signaling on fetal growth was examined by placental administration of cyclopamine, an SHH pathway inhibitor, to pregnant mice. Results The SHH pathway was downregulated in preeclampsia placentas, and its activation was highly correlated with birth weight. Trophoblast syncytialization was modulated by noncanonical SHH–adenylate cyclase (ADCY) signaling rather than canonical SHH–GLI signaling. The IGF1 receptor pathway was regulated by both noncanonical SHH–ADCY signaling and canonical SHH–GLI signaling. Inhibition of placental SHH signaling significantly reduced fetal weight in mice. Conclusion Placental development and fetal growth were regulated through the SHH pathway via the IGF axis.

Background: Poststroke depression (PSD) is the most frequent psychological sequela after stroke. Electroacupuncture (EA) treatment is effective for PSD. The study aimed at clarifying the mechanisms of EA's antidepressant effects in a PSD rat model. Methods: We used middle cerebral artery occlusion to establish the rat model of PSD. Tests of sucrose preference and locomotor activity were performed to examine depressive-like behaviors. We measured malondialdehyde, GSH, SOD, IL6, IL1β, TNFα, and 5HT with ELISA. The hippocampal Shh-signaling pathway was assessed by Western blot. Results: EA significantly decreased sucrose preference and locomotor activities of PSD rats, reduced IL6, TNFα, increased GSH, and upregulated 5HT, and also slightly reduced IL1β and malondialdehyde, all of which were measured with ELISA. The Shh-signaling pathway assessed by Western blotting was activated by EA. Those changes were inhibited by the Shh-pathway inhibitor cyclopamine. Conclusion: EA effectively alleviated depressive-like behaviors in PSD by suppressing inflammation and oxidative stress through activation of the Shh-signaling pathway.

In this Account we present the total syntheses of artemisinin (an important antimalarial agent) and cyclopamine (the first natural inhibitor of the hedgehog signaling pathway). Furthermore, we describe the design and development of a γ-butyrolactone as an inhibitor of Gcn5 (a histone N-acetyltransferase), discuss the discovery of hyperforin and guttiferon G as the first natural products acting as inhibitors of sirtuins, and present the design of inhibitors of sialic acid biosynthesis. The biomedical background and importance are also discussed. Finally, we present the discovery and development of methods for transesterification, IBX-mediated oxidations, reduction of several functional groups with LiBH4/Me3SiCl, as well as a process enabling the synthesis of kilogram amounts of D-isocitric acid and its transformation to valuable chiral derivatives.1 Artemisinin and Malaria2 Cyclopamine and Cyclops3 Sunflowers, Krebs Cycle, and Isocitric acid4 Histones and N-Acetyltransferase Gcn55 St. John’s Wort, Hippocrates, and Sirtuins6 Sialic Acid and Inhibitors of Its Biosynthesis7 Transesterification8 IBX Reloaded9 And Finally: A Small Mistake with a Big Impact10 Epilogue

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.

Combination chemotherapy is considered to be one of the most effective treatments for breast cancer by reducing the emergence of drug resistance. In this study, a novel drug delivery system based on bovine serum albumin nanoparticles (BSA NPs) was successfully developed. Doxorubicin (DOX) and cyclopamine (CYC), a potential anti-cancer agent that inhibits the hedgehog signaling pathway were entrapped into BSA NPs through electrostatic interactions and hydrophobic interactions, respectively. Rather than simple combination of two different chemotherapeutics, the CYC also increased the intracellular DOX accumulation by decreasing the expression of P-glycoprotein (P-gp), which could thus reverse the DOX resistance. Tumor-targeting property of nanoparticles was the prerequisite for its further application. Interestingly, retention of fluorescently-labeled particles in vivo indicated that the dual-drug-loaded BSA NPs could not only target the primary tumors, but also target the metastatic lymph nodes, which would simultaneously inhibit the tumor growth and distant metastasis. Taken together, this study provides a promising strategy for co-delivery of drugs, tumor and metastatic lymph node targeting, and DOX resistance reversing in breast cancer chemotherapy.

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.

Hedgehog (Hh) signaling is involved in early embryogenesis and maintains quiescence in the adult lungs. The interruption of Hh signaling may lead to the development of chronic obstructive pulmonary disease (COPD). The current study aimed to assess whether the Hh pathway affects cigarette-induced emphysema and airway inflammation by regulating inflammatory cytokines. C57BL/6J mice were randomized into control, cigarette smoke (CS) or CS + cyclopamine (CSC) groups. Control mice were exposed to normal room air, CS mice were exposed to tobacco smoke and CSC mice were exposed to CS and received cyclopamine treatment. Histopathological examination of lung tissues was performed, and the expression of sonic hedgehog (HH), glioma-associated oncogene homolog 1 (Gli1), hedgehog-interacting protein (HIP) and several inflammatory mediators (intracellular adhesion molecule-1, IL-6, IL-8 and TNF-α) were compared using reverse transcription-quantitative PCR and western blotting. The emphysema of lung tissues by histopathological examination demonstrated partial amelioration in the CSC group compared with that in the CS group. Additionally, expression levels of SHH, Gli1 and inflammatory mediators were significantly higher in the CS group compared with the control group but were significantly decreased in the CSC group. The expression of HIP was decreased in the CS group, but significantly increased in the CSC group. Hh signaling may serve an important role in cigarette-induced emphysema and airway inflammation by regulating inflammatory cytokines in animal models. Therefore, diminishing the activation of the Hh signal may serve as a novel therapeutic strategy for patients suffering from smoking-related COPD.

Benign prostatic hyperplasia (BPH) is a common disease in aging males. It has been proven that the Hedgehog (HH) is implied as an effective and fundamental regulatory growth factor signal for organogenesis, homeostasis, and regeneration. Smoothened (SMO), as the major control point of HH signals, activates aberrantly in most human solid tumors. However, the specific function of SMO and its downstream glioma-associated oncogene (GLI) family in BPH has not been well understood. Here, we first revealed that the SMO cascade was upregulated in BPH tissues and was localized in both the stromal and the epithelium compartments of human prostate tissues. Cyclopamine, as a specific SMO inhibitor, was incubated with BPH-1 and WPMY-1, and intraperitoneally injected into a BPH rat model established by castration with testosterone supplementation. SMO inhibition could induce cell apoptosis, cell cycle arrest at the G0/G1 phase, and a reduction of tissue fibrosis markers, both in vitro and in vivo. Finally, a tissue microarray, containing 104 BPH specimens, was constructed to analyze the correlations between the expression of SMO cascade and clinical parameters. The GLI2 was correlated positively with nocturia and negatively with fPSA. The GLI3 was in a positive relationship with International Prostate Symptom Score and nocturia. In conclusion, our study suggested that SMO cascade could play important roles in the development of BPH and it might be rediscovered as a promising therapeutic target for BPH.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening. While medial SMCs contribute, the participation of hedgehog responsive resident vascular stem cells (vSCs) to lesion formation remains unclear. Using transgenic eGFP mice and genetic lineage tracing of S100β vSCs in vivo, we identified S100β/Sca1 cells derived from a S100β non-SMC parent population within lesions that co-localise with smooth muscle ⍰-actin (SMA) cells following iatrogenic flow restriction, an effect attenuated following hedgehog inhibition with the smoothened inhibitor, cyclopamine. In vitro, S100β/Sca1 cells isolated from atheroprone regions of the mouse aorta expressed hedgehog signalling components, acquired the di-methylation of histone 3 lysine 4 (H3K4me2) stable SMC epigenetic mark at the Myh11 locus and underwent myogenic differentiation in response to recombinant sonic hedgehog (SHh). Both S100β and PTCH1 cells were present in human vessels while S100β cells were enriched in arteriosclerotic lesions. Recombinant SHh promoted myogenic differentiation of human induced pluripotent stem cell-derived S100β neuroectoderm progenitors In vitro. We conclude that hedgehog responsive S100β vSCs contribute to lesion formation and support targeting hedgehog signalling to treat subclinical arteriosclerosis.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a promising anti-myeloma drug prototype. The aim of the present study was to investigate the synergistic effects of cyclopamine and circularly permuted TRAIL (CPT) on the proliferation and apoptosis of multiple myeloma cells. The results showed that the inhibitory effects of cyclopamine on the proliferation of human myeloma RPMI-8226 and SKO-007 cells were weak. RPMI-8226 cells were sensitive to CPT; however, the proliferation of SKO-007 cells was not effectively inhibited by CPT. SKO-007 cells were thus considered resistant to cyclopamine and CPT and used for subsequent experiments. Treatment with a combination of cyclopamine and CPT significantly inhibited cell proliferation. Moreover, the Q value showed that cyclopamine combined with CPT could synergistically inhibit the proliferation of SKO-007 cells. Cyclopamine increased CPT-induced apoptosis in the SKO-007 cells and exhibited a synergistic induction of apoptosis when combined with CPT. Moreover, the combination of cyclopamine and CPT decreased the ratio of myeloma stem cells. Quantitative PCR showed that cyclopamine decreased the mRNA expression levels of GLI1/GLI2/GLI3 and increased the expression levels of death receptor 4. In conclusion, the present study showed that a combination of cyclopamine and CPT exhibited synergistic effects on the inhibition of proliferation and induction of apoptosis in myeloma cells.

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.

Background: Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective therapy. Blood-brain barrier (BBB) damage is a hallmark of ICH-induced brain injury that leads to edema formation, leukocytes infiltration, influx of blood components into the perihematomal (PHE) region, and eventually brain injury. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted molecules that contribute to the association between these cells. Sonic hedgehog (SHH) derived from astrocytes promotes the maturity and integrity of the BBB by upregulating tight junctions (TJs) in brain capillary endothelial cells (ECs). However, the effect of SHH on BBB in ICH has not been investigated.Methods: Cyclopamine (CYC) is a potent, selective inhibitor that specifically blocks the SHH signaling pathway. Here, we used pharmacological inhibitions (CYC and its derivatives) to determine a critical role of the SHH signaling pathway in promoting BBB integrity after ICH by mechanisms of regulating the TJ proteins in vivo and in vitro.Results: The expression of astrocytic SHH was upregulated in mouse brains after ICH. Compared with the vehicle-treated group, inhibition of the SHH signaling pathway with CYC and its derivatives treatments aggravated neurological function deficits, brain edema, hematoma volume, and BBB impairment by downregulating TJs in ECs through the SHH-Gli-1 axis in vivo and in vitro.Conclusions: SHH signaling pathway at the level of the BBB provides a barrier-promoting effect, suggesting that the SHH signaling pathway may function as a potential therapeutic target for restoring BBB function in ICH.

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.

Photodynamic therapy (PDT) has shown great promise in breast cancer treatment. However, simplex target ligand modification or stimuli release cannot meet the requirement of effective drug delivery to solid tumor tissue. To overcome continuous bio-barriers existing in the tumor microenvironment, multi-stage response drug delivery was desirable. Herein, we developed a unique tumor microenvironment tailored nanoplatform for chlorin e6 (Ce6) delivery. We chose bovine serum albumin (BSA) as “mother ships” material for effective tumor periphery resident, cyclopamine (CYC) as extracellular matrix (ECM) inhibitor and synergistic anti-tumor agent, and diselenide containing amphiphilic hyaluronic acid-chlorin e6 polymers (HA-SeSe-Ce6) synthesized as “small bombs” for internal tissue destruction. The above three distinct function compositions were integrated into an independent CYC and HA-SeSe-Ce6 co-delivery albumin nano-system (ABN@HA-SeSe-Ce6/CYC). The obtained nano-system presents good biocompatible, long circulation and effective tumor accumulation. After entering tumor microenvironment, CYC gradually releases to disrupt the ECM barrier to open the way for further penetration of HA-SeSe-Ce6. Subsequently, targeted tumor cell internalization and intracellular redox response release of Ce6 would achieve. Moreover, CYC could also make up the deficiency of Ce6 in hypoxia area, owing to its anti-tumor effect. Improved therapeutic efficacy was verified in a breast cancer cell line and tumor-bearing mice model.

Background: Intratumoral hypoxia contributes to cancer progression and poor prognosis. Carbonic anhydrases IX (CAIX) and XII (CAXII) play pivotal roles in tumor cell adaptation and survival, as aberrant Hedgehog (Hh) pathway does. In malignant melanoma both features have been investigated for years, but they have not been correlated before and/or identified as a potential pharmacological target. Here, for the first time, we demonstrated that malignant melanoma cell motility was impaired by targeting CAXII via either CAs inhibitors or through the inhibition of the Hh pathway. Methods: We tested cell motility in three melanoma cell lines (WM-35, SK-MEL28, and A375), with different invasiveness capabilities. To this end we performed a scratch assay in the presence of the smoothened (SMO) antagonist cyclopamine (cyclo) or CAs inhibitors under normoxia or hypoxia. Then, we analyzed the invasiveness potential in the cell lines which were more affected by cyclo and CAs inhibitors (SK-MEL28 and A375). Western blot was employed to assess the expression of the hypoxia inducible factor 1&alpha, CAXII, and FAK phosphorylation. Immunofluorescence staining was performed to verify the blockade of CAXII expression. Results: Hh inhibition reduced melanoma cell migration and CAXII expression under both normoxic and hypoxic conditions. Interestingly, basal CAXII expression was higher in the two more aggressive melanoma cell lines. Finally, a direct CAXII blockade impaired melanoma cell migration and invasion under hypoxia. This was associated with a decrease of FAK phosphorylation and metalloprotease activities. Conclusions: CAXII may be used as a target for melanoma treatment not only through its direct inhibition, but also through Hh blockade.

This study aimed to examine the effect of ubiquitin-specific peptidase 7 (USP7) on the proliferation and differentiation of ATDC5 cells and explore the underlying mechanisms. PCR, western blot, and immunofluorescence staining were used to observe the expression of USP7 after chondrogenic induction. The expressions of markers of chondrogenic and hypertrophic differentiation, and parathyroid hormone-related protein (PTHrP)/parathyroid hormone 1 receptor (PTH1R) signalling, were assessed by PCR, western blot, and histological staining under USP7 knockdown or its inhibitor. Cell proliferation was assessed by the CCK-8 assay and crystal violet staining. An in vivo experiment was performed to verify the functions of USP7 through histological and immunohistochemistry staining. Cyclopamine and abaloparatide were used to verify the signalling pathway. The interactions between USP7 and both PTHrP and sex-determining region Y-box 9 (Sox9) were tested by co-immunoprecipitation. The relationship between Sox9 and PTHrP was tested by chromatin immunoprecipitation and siRNA. USP7 knockdown or its inhibitor suppressed cell proliferation and chondrogenic differentiation but improved hypertrophic differentiation. The in vivo study obtained the same results. USP7 knockdown or its inhibitor inhibited PTHrP/PTH1R signalling to exert its function. Supplementation with cyclopamine suppressed PTHrP/PTH1R signalling and inhibited ATDC5 cell proliferation and differentiation. Supplementation with abaloparatide activated PTH1R to upregulate proliferation and chondrogenic differentiation but downregulated hypertrophic differentiation. Furthermore, USP7 interacted with Sox9 and Sox9 bound to PTTHrP to promote its expression. In conclusion, USP7 modulates the proliferation and differentiation of ATDC5 cells via the Sox9-PTHrP-PTH1R axis.

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.

PURPOSE To study the mechanism of vascular endothelial growth factor (VEGF) regulated by miR-375 and Sonic hedgehog (SHH) signaling pathways on the characteristics of trophoblast cells (TCs) in preeclampsia (PE) rats. METHODS Female (100) Wistar rats in rut were mated with males (50) at a ratio of 2:1, and 60 pregnant rats were assigned to a normal group (Nor, n=10) and a model group (Mod, n=50) in a random manner. Rat TCs of placental villus were isolated and were divided into 8 groups. The related indicators in each group were detected, respectively. RESULTS In contrast to the NC group, miR-375 expression greatly elevated in the miR-375 mimic group (mimic group); in the mimic and cyclopamine groups, VEGF, proliferating cell nuclear antigen（PCNA, N- cadherin and Bcl-2 expression, cell proliferation, S phase cells, and migration and invasion ability were significantly decreased and E-cadherin and Bax expression, G1 phase cells and apoptosis rate were increased significantly, but in Over expressing-VEGF（ov-VEGF） group, the above indicators presented contrary results (all P< 0.05). In contrast to the mimic group, the expression of SHH, VEGF, PCNA, N- cadherin and Bcl-2, cell proliferation, S phase cells and the invasion and migration activity in the miR-375 mimic + oe-VEGF group were evidently increased. CONCLUSIONS Over-expressing miR-375 can inhibit the expression of VEGF, thus slow down the invasion and proliferation of TCS in PE rats, which is realized by regulating SHH signal pathway.

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.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0&ndash, 48 h (early stage of osteoclast differentiation) or 48&ndash, 96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

Purmorphamine (PUR), an agonist of the Smoothened (Smo) receptor, has been shown to function as a neuroprotectant in acute experimental ischemic stroke. Its role in hypoxic-ischemic (HI) brain injury in neonatal mice remains unknown. Here we show that PUR attenuated acute brain injury, with a decrease in Bax/Bcl-2 ratio as well as inhibition of caspase-3 activation. These beneficial effects of PUR were associated with suppressing neuro-inflammation and oxidative stress. PUR exerted long-term protective effects upon tissue loss and improved neurobehavioral outcomes as determined at 14 and 28 days post-HI insult. Moreover, PUR increased synaptophysin (Syn) and postsynaptic density (PSD) protein 95 expression in HI-treated mice and attenuated synaptic loss. PUR upregulated the expression of Shh pathway mediators, while suppression of the Shh signaling pathway with cyclopamine (Cyc) reversed these beneficial effects of PUR on HI insult. Our study suggests a therapeutic potential for short-term PUR administration in HI-induced injury as a result of its capacity to exert multiple protective actions upon acute brain injury, long-term memory deficits, and impaired synapses. Moreover, we provide evidence indicating that one of the mechanisms underlying these beneficial effects of PUR involves activation of the Shh signaling pathway.

Traumatic brain injury (TBI) is a leading cause of death and disability globally. No drug treatments are available, so interest has turned to endogenous neural stem cells (NSCs) as alternative strategies for treatment. We hypothesized that regulation of cell proliferation through modulation of the sonic hedgehog pathway, a key NSC regulatory pathway, could lead to functional improvement. We assessed sonic hedgehog (Shh) protein levels in the cerebrospinal fluid (CSF) of patients with TBI. Using the cortical contusion injury (CCI) model in rodents, we used pharmacological modulators of Shh signaling to assess cell proliferation within the injured cortex using the marker 5-Ethynyl-2’-deoxyuridine (EdU); 50mg/mL. The phenotype of proliferating cells was determined and quantified. Motor function was assessed using the rotarod test. In patients with TBI there is a reduction of Shh protein in CSF compared with control patients. In rodents, following a severe CCI, quiescent cells become activated. Pharmacologically modulating the Shh signaling pathway leads to changes in the number of newly proliferating injury-induced cells. Upregulation of Shh signaling with Smoothened agonist (SAG) results in an increase of newly proliferating cells expressing glial fibrillary acidic protein (GFAP), whereas the Shh signaling inhibitor cyclopamine leads to a reduction. Some cells expressed doublecortin (DCX) but did not mature into neurons. The SAG-induced increase in proliferation is associated with improved recovery of motor function. Localized restoration of Shh in the injured rodent brain, via increased Shh signaling, has the potential to sustain endogenous cell proliferation and the mitigation of TBI-induced motor deficits albeit without the neuronal differentiation.

Background: The chronic fibrosis and hypertrophy of ligamentum flavum (LF) is an important cause of lumbar spinal canal stenosis (LSCS). Our previous work showed WNT1 inducible signaling pathway protein 1 (WISP-1) is a critical driver of LF fibrosis. However, its potential mechanism has not been explored. Methods: We studied the expression of Hedgehog-related proteins in human LF tissues. Cell viability, cell cycle, apoptosis rate and molecular mechanisms were evaluated in LF cells with lentivirus-mediated knockdown or over-expression of WISP-1 and Gli1. Finally, the molecular mechanism was confirmed in in vivo studies. Findings: The results showed Gli1 was upregulated in hypertrophic LF tissues and required for fibroblast proliferation, apoptosis inhibition and collagen expression in fibroblasts. Moreover, mechanical stretching increased the expression of WISP-1 in LF fibroblasts. Furthermore, WISP-1 induced fibrogenesis in vitro through Hedgehog-Gli1 pathway. This conclusion was supported by the fact that WISP-1 activated Hedgehog-Gli1 pathway in LF fibroblasts and cyclopamine attenuated the effect of WISP-1-induced fibrogenesis. WISP-1 also promoted the transition of fibroblasts into myofibroblasts via Hedgehog pathway. Importantly, hypertrophic LF rabbit model induced by mechanical stress also showed pathological change of fibrosis and more higher expression of WISP-1, Gli1 and ɑ-SMA. Therapeutic administration of cyclopamine reduced collagen expression, fibroblast proliferation, myofibroblast differentiation and ameliorated fibrosis in rabbit model of mechanical stress-induced LF fibrosis. Interpretation: Our study showed the mechanical stress/WISP-1/Hedgehog signaling as a new fibrotic axis contributing to the LF hypertrophy and identified Hedgehog signaling as a therapeutic target for the prevention and treatment of LF fibrosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant No. 81802198) and Natural Science Foundation of Jiangsu Province (Grant No. SBK2019022658). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: An informed consent form was signed by the all patients and the study was approved by the Ethics Committee of Nanjing Medical University (Approval No.20180104).

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure-activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine-based delivery approaches for Hh pathway inhibitors are also discussed concisely.

Background Inflammation, oxidative stress and epithelial barrier dysfunction have been implicated in inflammatory bowel disease (IBD) pathology. The targeted inhibition of these features may represent a promising therapeutic strategy for IBD. Polydatin is an effective natural antioxidant that possesses strong antioxidant and anti-apoptotic properties. Thus, we studied the protective effects of polydatin treatments on a mouse model of experimental colitis. Methods Acute colitis was experimentally induced by adding 3% dextran sulfate sodium (DSS) to the drinking water provided to mice for 7 days and by administering different doses of polydatin (15, 30, or 45 mg/kg) during the same period. Mice were also treated with the Sonic hedgehog (Shh) pathway inhibitor cyclopamine to estimate the efficacy of polydatin and Shh inhibitors on colitis. The disease activity index (DAI), colon length, histology, levels of oxidative and apoptotic mediators and levels of Shh pathway components were evaluated. Results The polydatin treatment significantly attenuated the DAI, colon shortening and histological damage. In addition, polydatin administration effectively decreased malondialdehyde (MDA) levels and increased the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Polydatin also inhibited apoptosis in mice with colitis by downregulating the expression of the pro-apoptotic proteins Bax, caspase 3 and cleaved caspase 3 and increasing the expression of the anti-apoptotic protein Bcl-2. Furthermore, polydatin modulated Shh signaling pathway activation. After polydatin treatment, the main components of the Shh pathway, including Shh, Patched (Ptc), Smoothened (Smo), and glioblastoma-1 (Gli1), were upregulated at the mRNA and protein levels. Blockade of the Shh pathway using cyclopamine abolished the effects of polydatin on mice with colitis. Conclusion Based on these observations, polydatin may suppress experimental colitis at least partially by regulating the Shh signaling pathway.

Phosphatidylethanolamine-binding protein 4 (PEBP4), a member of the PEBP family, has been reported to play a pivotal role in tumor progression. However, its role in epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells remains unclear. Here, we investigated the effects and underlying mechanism of PEBP4 in NSCLC EMT. Three human NSCLC cell lines (A549, H1299, and H460) were transfected with pcDNA3.1-PEBP4 or PEBP4-targeting small interfering RNA. Then, cell proliferation was analyzed by the MTT assay, and cell migration and invasion were analyzed by the transwell chamber assay. Protein and messenger RNA expression of the related genes and proteins were assessed by Western blot analysis and quantitative real-time polymerase chain reaction, respectively. Results showed that PEBP4 was highly expressed in the human lung cancer tissues and three human NSCLC cell lines. Pretreatment with pcDNA3.1-PEBP4 promoted the proliferation, invasion, and migration of NSCLC cells and increased EMT in vitro and lung tumor metastasis in vivo. Whereas knockdown of PEBP4 suppressed NSCLC cell migration, PEBP4, and invasion with prevented EMT. Furthermore, PEBP4 overexpression significantly promoted the transcriptional activity of sonic hedgehog (Shh) signaling in NSCLC cells. Further analysis showed that using cyclopamine to inhibit Shh signaling significantly ameliorated the effect on cell proliferation, invasion, and migration, as well as EMT triggered by PEBP4 overexpression. Together, these results suggest that PEBP4 may promote tumorigenesis in NSCLC by regulating cell proliferation and EMT via activation of the Shh signaling pathway.

Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease.