Shh Expression Research Articles

A murine model of large-scale bone regeneration reveals a selective requirement for Sonic Hedgehog

Building and maintaining skeletal tissue requires the activity of skeletal stem and progenitor cells (SSPCs). Following injury, local pools of these SSPCs become active and coordinate to build new cartilage and bone tissues. While recent studies have identified specific markers for these SSPCs, how they become activated in different injury contexts is not well-understood. Here, using a model of large-scale rib bone regeneration in mice, we demonstrate that the growth factor, Sonic Hedgehog (SHH), is an early and essential driver of large-scale bone healing. Shh expression is broadly upregulated in the first few days following rib bone resection, and conditional knockout of Shh at early but not late post-injury stages severely inhibits cartilage callus formation and later bone regeneration. Whereas Smoothened (Smo), a key transmembrane component of the Hh pathway, is required in Sox9+ lineage cells for rib regeneration, we find that Shh is required in a Prrx1-expressing, Sox9-negative mesenchymal population. Intriguingly, upregulation of Shh expression and requirements for Shh and Smo may be unique to large-scale injuries, as they are dispensable for both complete rib and femur fracture repair. In addition, single-cell RNA sequencing of callus tissue from animals with deficient Hedgehog signaling reveals a depletion of Cxcl12-expressing cells, which may indicate failed recruitment of Cxcl12-expressing SSPCs during the regenerative response. These results reveal a mechanism by which Shh expression in the local injury environment unleashes large-scale regenerative abilities in the murine rib.

Study on the Mechanism of Sancao Tiaowei Decoction in the Treatment of MNNG-Induced Precancerous Lesions of Gastric Carcinoma Through Hedgehog Signaling Pathway.

Sancao Tiaowei Decoction (SCTWD), a traditional Chinese medicine created by Professor Chen Weijian, has been used in the prevention and treatment of precancerous lesions of gastric carcinoma (PLGC). However, its mechanism has not been made clear. This study aimed to evaluate the therapeutic effect of SCTWD on 1-methyl-3-nitro-1-nitrosoguanidine-induced PLGC in rats and the mechanism of this effect. We found that SCTWD effectively repaired gastric mucosal injury, reversed the process of PLGC, and inhibited the occurrence of gastric cancer to some extent. In the results of hematoxylin-eosin (HE) staining, the number and arrangement of mucosal glands and the number of mononuclear cells in the lamina propria were improved in varying degrees; the enzyme-linked immunosorbent assay (ELISA) showed that the PG I and PGR of the medication treatment group were significantly higher; a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) test showed that SCTWD could significantly upregulate the expression levels of Shh, Ptch, and Gli-1 in the gastric tissue of rats. The immunohistochemical method showed that SCTWD could significantly upregulate the protein expressions of Shh, Gli-1, Smo, cyclin D1, CDKN2A/p16INK4a, and NF-κBP65 and could reduce the expression of Ptch at the same time. Through the preliminary analysis of 75 compounds screened by UPLC-Q-TOF-MS, the main components, such as organic acids, esters and anhydrides, flavonoids, phenols, tanshinones, and so on, have anti-inflammatory and anti-tumor pharmacological effects. The results of KEGG enrichment analysis showed that 5 signaling pathways related to this project were found, and 33 differential genes were presented to construct the interaction network. These results suggested that SCTWD had a good regulatory effect on PLGC and thus may have a multi-targeted effect; SCTWD can not only significantly improve the pathological changes of gastric mucosa in rats with PLGC but also exert a strong effect of the regulation of the hedgehog signaling pathway.

An E‐Box Binding Site is Sufficient for ZPA Regulatory Sequence (ZRS) Activity in the Limb

The zone of polarizing activity (ZPA) is a cluster of mesodermal cells that secrete Sonic hedgehog (Shh) to regulate anterior‐posterior patterning during limb development. The ZPA regulatory sequence (ZRS) is the limb‐specific enhancer that controls Shh expression. ZRS microdeletion abrogates Shh expression and posterior limb development, whereas ZRS single nucleotide variants (SNVs) produce anterior ectopic Shh and preaxial polydactyly. However, the ZRS mechanism of action is not well characterized. Transcription factors (TFs) Hand2, Twist1, and Hoxd13 regulate Shh expression and ZRS contains putative binding sites for each: two Hoxd13 binding sites and three E‐boxes, sites targeted by basic helix‐loop‐helix (bHLH) TFs such as Hand2 and Twist1. Further, reports suggest these TFs may act through the ZRS: Hand2 purportedly binds the ZRS through a central E‐box; as bHLH‐TFs Hand2 and Twist1 may regulate ZRS as homo‐ or heterodimers; and both Hand2 and Hoxd13 activate the ZRS in vitro. Previously we demonstrated that disrupting all five binding sites eliminates ZRS activity. In this report, we further investigate these sites to determine the contribution of each to ZRS activity. We hypothesized that the Hand2 binding site is required for activity and that other E‐boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm.We assessed ZRS activity via an enhancer‐GFP reporter, altering all five binding sites to eliminate activity, then reinstating each site individually using site‐directed mutagenesis. Constructs were electroporated into the presumptive limbs of chicken embryos (Hamburger‐Hamilton stage 14). Enhancer activity was evaluated 48 hours post‐electroporation by fluorescence microscopy. Restoring either Hoxd13 site led to weak, focal activity. Restoring two of the E‐boxes had little to no effect, however, restoring the third, 3’ E‐box was sufficient for robust activity. This was unexpected since the central E‐box was reported as the likely Hand2 binding site. Our data suggest that the 3’ E‐box is likely the critical site used by Hand2 in ZRS‐mediated Shh regulation. Additionally, the Hoxd13 binding sites appear to impart focal activity supporting a role for localization. These findings further characterize the mechanism used to regulate focal Shh expression during limb development.

LHX2 Regulates Shh Expression in the Limb Independent of the ZPA Regulatory Sequence

During limb development, proximal‐distal patterning is controlled by fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER), while anterior‐posterior expansion is coordinated by sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA). Both FGFs and SHH regulate each other through an autoregulatory loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate of the Fgf‐to‐Shh portion of the loop. In the absence of Lhx2 (and its redundant family member, Lhx9), Shh expression is markedly reduced. However, the mechanism by which LHX2 mediates FGF‐regulation of Shh is unclear. Shh is regulated by a limb‐specific enhancer called the ZPA Regulatory Sequence (ZRS). Using in silicoanalysis, our lab has identified two putative LHX2 binding sites within the ZRS. We hypothesized that LHX2 regulates Shh expression directly via the ZRS. To determine if LHX2 binding sites were necessary for ZRS activity, we mutated both LHX2 binding sites within a control tk‐ZRS‐eGFP reporter construct using site‐directed mutagenesis. We electroporated these constructs into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14) and assessed for changes in ZRS activity in the limb buds 48 hours post transfection via fluorescence microscopy. We found that the mutation of both LHX2 binding sites within the ZRS resulted in activity similar to our control. These results suggest that LHX2 may not bind directly to the ZRS in its regulation of Shh. Instead, LHX2 may use another pathway to regulate Shhexpression. Further experiments are needed to confirm this connection. Investigating the mechanism by which LHX2 regulates Shh will provide insight into how LHX2 contributes to coordinated patterned‐limb development.

Regulation of LIM homeodomain 2 (Lhx2) in the distal limb bud during patterning and development

During limb morphogenesis, fibroblast growth factors (Fgfs) secreted from the apical ectodermal ridge (AER) direct proximodistal outgrowth of the limb, while sonic hedgehog (Shh) released from the zone of polarizing activity modulate anteroposterior expansion of the limb. Fgfs and Shh maintain each other’s expression through an autoregulatory loop. The transcription factor LIM homeodomain 2 (Lhx2) is expressed in the distal sub‐AER mesoderm and is important for Shh expression. We have identified a chicken enhancer upstream of the LHX2 promoter that is active in the distal sub‐AER LHX2 expression domain that contains putative transcription factor binding sites for known downstream targets of FGF signaling that are critical for activity. Therefore, we hypothesized that LASARM1 interacts with the LHX2 promoter and that FGF works through LASARM1 to regulate LHX2 expression. To determine this, we performed chromosome conformation capture (3C) on cells isolated from distal chicken limb buds and compared them to non‐LHX2 expressing tail cells. Promoter and enhancer specific primers were used to isolate ligated promoter‐enhancer fragments by end‐point PCR. Amplified sequences were then sequence validated. A 3C ligation product library was used as a control. We also evaluated the role of FGF to regulate LASARM1 activity in subAER mesoderm by analyzing LASARM1 activity following inhibition of FGF signaling by an FGF‐receptor inhibitor. End‐point PCR revealed promoter‐enhancer ligated amplicons in distal limb bud cells, but not in tail cells. DNA sequencing of enhancer‐ligated fragments confirmed LHX2‐LASARM1 fusion fragments for distal limb bud cells. Interestingly, disruption of FGF signaling via a specific FGF receptor inhibitor did not alter LASARM1 activity, although LHX2 expression was markedly reduced. These findings suggest that LASARM1 interacts with the LHX2 promoter during limb patterning, but additional enhancers are required to mediate the FGF‐mediated up‐regulation of LHX2. Evaluation of additional LHX2 enhancers is underway. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying LHX2 regulation and the FGF‐to‐SHH reciprocal loop.

Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration.

After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.

CGRP and Shh Mediate the Dental Pulp Cell Response to Neuron Stimulation

Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.

C/EBP homologous protein promotes Sonic Hedgehog secretion from type II alveolar epithelial cells and activates Hedgehog signaling pathway of fibroblast in pulmonary fibrosis

BackgroundEndoplasmic reticulum (ER) stress is involved in the pathological process of pulmonary fibrosis, including IPF. It affects a broad scope of cellular types during pulmonary fibrosis but the role in epithelial-mesenchymal crosstalk has not been fully defined. The present study aimed to investigate the effects of Shh secretion by ER stress-challenged type II alveolar epithelial cells (AECII) on fibroblast and pulmonary fibrosis.MethodsConditioned medium (CM) from tunicamycin (TM)-treated AECII was collected and incubated with fibroblast. Short hairpin RNA (shRNA) was used for RNA interference of C/EBP homologous protein (CHOP). The effects of CHOP and HH signaling were evaluated by TM administration under the background of bleomycin-induced pulmonary fibrosis in mice.ResultsBoth expression of CHOP and Shh in AECII, and HH signaling in mesenchyme were upregulated in IPF lung. TM-induced Shh secretion from AECII activates HH signaling and promotes pro-fibrotic effects of fibroblast. Interfering CHOP expression reduced ER stress-induced Shh secretion and alleviated pulmonary fibrosis in mice.ConclusionsOur work identified a novel mechanism by which ER stress is involved in pulmonary fibrosis. Inhibition of ER stress or CHOP in epithelial cells alleviated pulmonary fibrosis by suppressing Shh/HH signaling pathway of fibroblasts.

An early Shh-H2O2 reciprocal regulatory interaction controls the regenerative program during zebrafish fin regeneration.

Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Hydrogen peroxide (H2O2), the major ROS produced by cells, acts as a second messenger to modify redox-sensitive proteins or lipids. After caudal fin amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways remain poorly understood. We focused on the early dialog between H2O2 and Sonic hedgehog (Shh) during zebrafish fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, the means of this tight reciprocal control change during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of an early positive crosstalk between Shh and H2O2 that might be more generally involved in various processes paving the way to improve regenerative processes, particularly in vertebrates.

Differential promoter usages of PTCH1 and down regulation of HHIP are associated with HNSCC progression

PurposeThe study was aimed to understand the importance of the hedgehog signaling pathway in development of head and neck squamous cell carcinoma (HNSCC). MethodsThe molecular profiles of the key regulatory genes of the pathway were analysed in the adjacent normal epithelium and tumor samples. The findings were validated in HNSCC cell line. ResultsIn the bioinformatical analysis, severe reduction in the expression of HHIP was evident in the datasets. The protein and mRNA expression studies in our sample pool revealed interplay of various isoforms of PTCH1 gene (PTCH1–1 and 1B) together with high/medium expression of GLI, SHH, SMO and HHIP in the basal/parabasal layers of the normal epithelium. As the disease progressed, severe downregulation of HHIP coupled with upregulation of GLI1 and differential expression pattern of various PTCH1 gene isoform was evident. Promoter methylation analysis of PTCH1 gene revealed the involvement of more than one promoter of PTCH1 in regulating the expression of different isoform of this gene during tumorigenesis. Treating the FaDu cell line with the demethylating agent 5-aza-2′-deoxycytidine reversed the methylation effects of HHIP and PTCH1 and de-activated the pathway. Also, reduced expression of HHIP-AS1 was observed in our sample pool suggesting multiple ways of regulation of the HHIP gene. Lastly, the patients with under expression of HHIP, HHIP-AS1, high expression of GLI1 showed worse five-year over-all survival trend. ConclusionDynamic promoter switching of PTCH1 and frequent inactivation of HHIP are the key regulatory events of hedgehog pathway activation in HNSCC.

The Overexpression of Sonic Hedgehog Associates with Collateral Development and Amelioration of Oxidative Stress in Stroke Patients

PurposeSonic hedgehog (SHH) signaling pathway in oxidative stress condition has been acknowledged as a key trigger for angiogenesis and collateral vessel growth in the ischemic brain, and it exerts a protective effect on neuronal cells during oxidative stress. MethodsA total of sixty patients (n = 30 good collateral profile and n = 30 poor collateral profile) diagnosed with acute cerebral ischemia were enrolled in this study. qRT-PCR was performed to analyze the expression levels of SHH, Gli1, and superoxide dismutase (SOD), genes. Also, the serum levels of oxidative stress markers were determined in experimental groups. ResultsThe expression levels of SHH and Gli1 genes were significantly (p < 0.05) higher in stroke patients with good collateral circulation compared with those with poor collateral circulation, while SOD gene expression was similar between two groups (p > 0.05). A significantly positive correlation was found between the gene expression of SHH and Gli1 (r = 0.604, p < 0.001), SOD and Gli1 (r = 0.372, p < 0.003) genes. Our findings showed that the serum level of total antioxidant capacity (TAC) and Glutathione (GSH) and SOD enzyme activity was significantly (p < 0.05) increased, while serum total oxidant status (TOS) and malondialdehyde (MDA) levels were significantly (p < 0.05) decreased in patients with good collateral circulation as compared with those with poor collateral circulation. ConclusionOur observations shed light on the association of the SHH/Gli1 signaling pathway with cerebral collateral vessel development following ischemia. Oxidative stress in stroke patients with poor collateral circulation may result in the overexpression of SHH/Gli1 signaling pathway which possibly contribute to oxidative stress attenuation, as well as modulate angiogenesis and collateral vessels development.

Two nonsense GLI3 variants are associated with polydactyly and syndactyly in two families by affecting the sonic hedgehog signaling pathway.

BackgroundPolydactyly and syndactyly are congenital limb deformities, segregating in an autosomal‐dominant fashion. The variants in the GLI3 gene are closely related to congenital limb malformations. However, the causes underlying polydactyly and syndactyly are not well understood.MethodsWe conducted a whole‐exome sequencing on two four‐generation Chinese families with polydactyly and syndactyly. Then c.2374C>T and c.1728C>A mutant plasmids were transfected to HEK293T cells and mice limb bud cells to explore the functional consequences of these variants. Western blot and real‐time quantitative PCR were used to analyze the expression of GLI3 and Shh.ResultsIn these two families, the known GLI3 variant (NM_000168.6:c.2374C>T) and the novel GLI3 variant (NM_000168.6:c.1728C>A) contributed to polydactyly and syndactyly. Additionally, the GLI3 c.2374C>T mutant plasmid led to truncated GLI3 protein, and the GLI3 c.1728C>A mutant plasmid led to degraded GLI3 protein. Simultaneously, we demonstrated that the GLI3‐mutant plasmids led to decreased Shh expression in mice limb bud cells.ConclusionWe demonstrated that the novel GLI3 variant (c.1728C>A) and known GLI3 variant (c.2374C>T) contributed to the malformations in two four‐generation pedigrees with polydactyly and syndactyly by affecting SHH signaling.

Expression and significance of GLI1 and Shh in the malignant transformation of ovarian endometriosis

Objective: To investigate the expression levels and clinical significance of glioma-associated oncogene homolog 1 (GLI1) and sonic hedgehog signaling molecule (Shh) in the malignant transformation of ovarian endometriosis (EM). Methods: The expressions of GLI1 and Shh were detected by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and EnVision method in 50 cases of ovarian EM tissues, 35 cases of atypical endometriosis (aEM) and 50 cases of endometriosis-associated ovarian cancer (EAOC). The expression differences of two molecular markers in the malignant transformation of ovarian EM were compared, and the relationships between two molecular markers and the clinicopathological features and prognosis of EAOC were analyzed. Results: (1) RT-PCR showed that the expression levels of GLI1 mRNA in EM, aEM and EAOC group were 1.77±0.40, 3.54±0.44, and 7.80±0.24, respectively. The expression levels of Shh mRNA were 0.95±0.21, 3.14±0.35, and 5.41±0.31, respectively. GLI1 and Shh mRNA in EAOC group were significantly higher than those in EM and aEM group (all P<0.01), and there were statistically significant differences between EM and aEM group (all P<0.01). The percentages of GLI1 in ovarian EM, aEM and EAOC were 32% (16/50), 57% (20/35), and 66% (33/50), respectively, meanwhile, the positive expression rates of Shh were 20% (10/50), 49% (17/35), and 54% (27/50), respectively (all P<0.01). GLI1 mRNA expression was positively correlated with Shh mRNA expression in EAOC tissues (r=0.721, P<0.01). The expressions of GLI1 protein were proportionated to Shh protein in EAOC tissues (r=0.608,P=0.001). (2) The expression of GLI1 was significantly related to the International Federation of Gynecology and Obstetrics (FIGO) stage, cancer antigen 125 (CA125) levels, lymph node metastasis, and Platinum resistance in EAOC patients (all P<0.05). The expression of Shh were related to FIGO stage and lymph node metastasis in EAOC patients (all P<0.05). Logistic regression analysis showed that GLI1 expression was an independent risk factor for poor prognosis in EAOC patients (P<0.05). Kaplan-meier survival analysis showed that the overall survival rate of EAOC patients with high GLI1 expression and low GLI1 expression was 12.1% and 35.3%, respectively, with statistical significance (χ²=10.73, P<0.01). The overall survival rate of EAOC patients with high and low expression of Shh protein was 11.1% and 30.4%, in which there was statistically significant difference (χ²=3.96, P=0.047). Conclusion: GLI1 and Shh are highly associated with the malignant transformation of ovarian EM, which may play a role in promoting malignant degeneration of ovarian EM, and the high expression of GLI1 and Shh indicates a poor prognosis in EAOC patients.

Transforming growth factor-β1 induces transformation of rat meningeal fibroblasts into myofibroblasts by upregulating Shh signaling

To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts. Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR. TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05). TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.

Shen-Shuai-Ling Formulation Attenuates Renal Interstitial Fibrosis in Chronic Kidney Disease by Regulating SHH-Gli1 Signaling Pathway.

Background Shen-Shuai-Ling Formulation (SSLF) has apparent effects on improving renal function, delaying the progression of chronic kidney disease (CKD). Methods Fifty male SD rats were randomly divided into 5 groups: Sham group, Model group, SSLF group, CPN group, and C + S group. The morphological changes and the collagen fibers of the rat kidneys were observed by HE staining. The expression of α-SMA, Col I, SHH, Gli1, and snail1 was detected by Western blot and qPCR. Then, the cells were divided into the control group, SHH group, and SHH + SSLF serum group. Results Compared with the Model group, the fibrosis in SSLF, CPN, and C + S groups was significantly alleviated. And, compared with those in the Model group, the expression of α-SMA, Col I, SHH, Gli1, Snail in SSLF, CPN, and C + S groups decreased remarkably. Conclusions SSLF remarkably improves renal function and alleviates renal interstitial fibrosis in UUO rats.

Mesenchymal Stem Cells Inhibits the Shh/GLi (Sonic Hedgehog/GLi) Axis and Promotes Repair of Tissue Injury in Severe Acute Pancreatitis

Mesenchymal stem cells (MSCs) are indicated to severe pancreatitis (SAP), whilst level of Shh/GLi axis varies in severe acute pancreatitis (SAP). However, little is known the interaction between MSCs and Shh in SAP. In this study, we established animal model of SAP in 10 rats and transplanted MSCs into 10 rats, with another 10 sham-operated rats as control group. The pathological changes of rat pancreatic tissue were observed. ELISA was conducted to determine the MPO level of pancreatic inflammation, and Western blot to detect the expression level of Shh, Gli1 and Gli2 in tissues. Administration of MSCs remarkably alleviated the pancreatic tissue necrosis and inflammation and decreased blood loss in SAP rats. Up-regulated expression of Shh, Gli1 and Gli2 was observed in SAP tissues when compared to tissues in control group, but their expressions declined in the presence of MSCs, and 24 hour later returned to normal levels. Collectively, MSCs regulates the balance of Shh/GLi axis by decreasing Shh and Gli1, thereby attenuating progression and symptoms of SAP.

Improved Urothelial Cell Proliferation, Cytoskeleton and Barrier Function Protein Expression in the Patients With Interstitial Cystitis/Bladder Pain Syndrome After Intravesical Platelet-Rich Plasma Injection.

PurposeTo investigate urothelial cell proliferation, cytoskeleton, inflammation, and barrier function protein expressions in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) after intravesical platelet-rich plasma (PRP) injectionsMethodsA total of 19 patients with IC/BPS underwent 4 monthly intravesical PRP injections. Bladder biopsies were taken at the first and fourth PRP treatment. The bladder specimens were analyzed using the Western blot and immunochemical staining for progenitor cell markers for sonic hedgehog (Shh), CD34, and cytoskeleton proteins cytokeratin 5 (CK5), CK14, CK20; barrier function markers for zonula occludens-1 (ZO-1), E-cadherin, and intercellular adhesive molecule-1, tryptase and transforming growth factor-β (TGF-β). Global response assessment (GRA) was used to evaluate treatment outcomes.ResultsThe mean age of patients was 55.6 years. After PRP injections, the functional bladder capacity and maximum flow rate increased, and the visual analogue scale (VAS) of pain, interstitial cystitis (IC) symptom index, IC problem index, O’Leary-Sant symptom score, and GRA improved in all patients. Urothelium Shh, CK5, ZO-1, E-cadherin, and TGF-β expressions increased significantly after repeated PRP injections. By subgrouping, according to PRP treatment outcomes, significant increases in Shh, E-cadherin, and ZO-1 expressions were noted only in patients with GRA ≥1 or improved VAS, but not in patients with GRA=0 and no improvement in VAS.ConclusionsThe level of urothelial barrier function protein and cell proliferation protein expression in the patients with IC/BPS was increased after repeat intravesical PRP injections. Intravesical repeat PRP injections may have potential to improve urothelial health and result in symptoms improvement in the patients with IC/BPS.

High-fat diet intake ameliorates the expression of hedgehog signaling pathway in adult rat liver.

Disproportionate fatty diet intake provokes hepatic lipid accumulation that causes non-alcoholic fatty liver disease, triggering the embryonically conserved Hedgehog (Hh) pathway in the adult liver. The present study incorporates exploring the impact of chronically administered unsaturated (D-1) and saturated (D-2) fat-enriched diets on hematological parameters, liver functioning, and lipid profile in the rat model. Besides, hepatohistology and real time gene expression analysis of Hh signaling pathway genes i.e., Shh, Ihh, Hhip, Ptch1, Smo, Gli1, Gli2, and Gli3 were carried out. Fifteen Rattus norvegicus (♂) of 200 ± 25g weight were grouped into control, D-1, and D-2. Animals were fed on their respective diets for 16weeks. Fatty diet intake resulted in neutropenia, lymphocytosis, monocytosis, polycythemia, and macrocytosis in both experimental groups. Altered liver injury biomarkers, hypertriglyceridemia, and significantly increased very-low-density lipoprotein VLDL were also noted in both high-fat diet (HFD) groups as compared to control. Hepatohistological examination showed disrupted microarchitecture, infiltration of inflammatory cells, cellular necrosis, widened sinusoidal spaces, and microvesicular steatotic hepatocytes in D-1 and D-2. Collagen deposition in both HFD groups marks the extent of fibrosis. Significant upregulation of hedgehog pathway genes was found in fatty diet groups. In comparison with the control group, Shh Ihh, Hhip, Ptch1, Smo, Gli1, Gli2, and Gli3 were upregulated in D-1. In D-2 Shh, Hhip, and Smo expressions were upregulated, Ihh exhibited downregulation as compared to control. Excess fat deposits in liver due to chronic consumption of high-fat diet results in anomalous architecture and functioning. High-fat diet induced significant variations in Hh pathway genes expression; especially Shh, Ihh, Hhip, Ptch1, Smo, Gli1, Gli2, and Gli3 were upregulated. Infiltration of inflammatory cells ( ), widened sinusoidal spaces (▲), cellular necrosis, and micro vesicular steatotic hepatocytes (*) were shown in the liver. Significant collagen deposition in both HFD groups i.e. D-1 and D-2 confirmed liver fibrosis. Excessive intake of dietary fats impaired normal liver functioning and liver inflammation triggered Hh signaling in adult rats.

Pharmacological activation of the Sonic hedgehog pathway with a Smoothened small molecule agonist ameliorates the severity of alcohol-induced morphological and behavioral birth defects in a zebrafish model of fetal alcohol spectrum disorder.

Ethanol exposure during the early stages of embryonic development can lead to a range of morphological and behavioral differences termed fetal alcohol spectrum disorders (FASDs). In a zebrafish model, we have shown that acute ethanol exposure at 8-10hr postfertilization (hpf), a critical time of development, produces birth defects similar to those clinically characterized in FASD. Dysregulation of the Sonic hedgehog (Shh) pathway has been implicated as a molecular basis for many of the birth defects caused by prenatal alcohol exposure. We observed in zebrafish embryos that shh expression was significantly decreased by ethanol exposure at 8-10 hpf, while smo expression was much less affected. Treatment of zebrafish embryos with SAG or purmorphamine, small molecule Smoothened agonists that activate Shh signaling, ameliorated the severity of ethanol-induced developmental malformations including altered eye size and midline brain development. Furthermore, this rescue effect of Smo activation was dose dependent and occurred primarily when treatment was given after ethanol exposure. Markers of Shh signaling (gli1/2) and eye development (pax6a) were restored in embryos treated with SAG post-ethanol exposure. Since embryonic ethanol exposure has been shown to produce later-life neurobehavioral impairments, juvenile zebrafish were examined in the novel tank diving test. Our results further demonstrated that in zebrafish embryos exposed to ethanol, SAG treatment was able to mitigate long-term neurodevelopmental impairments related to anxiety and risk-taking behavior. Our results indicate that pharmacological activation of the Shh pathway at specific developmental timing markedly diminishes the severity of alcohol-induced birth defects.

Expression of Shh, Gli1, and Cyr61 in Gastric Cancer Predicts Overall Survival of Patients: A Retrospective Study.

This study aimed to evaluate the expression levels of Shh, Gli1, and Cyr61 proteins in gastric cancer tissues and analyze the relationship between these three proteins and the clinicopathological factors and prognosis of patients. This was a retrospective study. Four hundred gastric cancer tissue specimens from patients who underwent radical gastrectomy in Zhangye People's Hospital affiliated to Hexi University between February 2013 and February 2021 underwent immunohistochemical analysis. The positive expression rates of Shh, Gli1, and Cyr61 in gastric cancer tissues were 55.5%, 56.5%, and 64.5%, respectively. The expressions of Shh, Gli1, and Cyr61 in gastric cancer tissues were significantly correlated with tumor size, depth of invasion, and degree of differentiation (P < .05). The expression of Shh protein was positively correlated with the expression of Gli1 protein (P < .01), and the expression of Gli1 protein was positively correlated with the expression of Cyr61 protein (P < .01). Univariate and multivariate analyses showed that the expression of Shh, Gli1, and Cyr61 could predict the prognosis of patients (P < .05). Receiver operating characteristic curve analysis combined with TNM staging could better predict the three-year overall survival of patients (P < .05). Shh, Gli1, and Cyr61 proteins are significantly expressed in gastric cancer tissues and are risk factors for the prognosis of patients with gastric cancer.