Effect study of Sonic hedgehog overexpressed hair follicle stem cells in hair follicle regeneration / 中国修复重建外科杂志

OBJECTIVE@#To determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration.@*METHODS@#Hair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups.@*RESULTS@#The isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences ( P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant ( P<0.05).@*CONCLUSION@#Lentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.

O papel de proteínas das vias WNT/ß-Catenina, TGF-ß/BMP4 e SHH na odontogênese e odontomas / The role of proteins from the WNT/ß-Catenin, TGF-ß/BMP4 and SHH pathways in odontogenesis and odontomas

O desenvolvimento do dente depende de uma série de interações sinalizadoras recíprocas entre o epitélio oral (EO) e o ectomesênquima derivado da crista neural, a via WNT com o TGF-ß e BMP4 tem sido implicada na tumorigênese. A via de sinalização tipo Wingless (Wnt) / ß-catenina é essencial para a ativação precoce da odontogênese e no desenvolvimento de tumores odontogênicos. O TGF-ß e as BMPs tem sido associadas aos processos de dentinogênese reacionária e reparadora. A sinalização de Shh pode regular a proliferação celular no ectomesênquima dentário, controlando assim a morfogênese dentária. O objetivo da pesquisa foi investigar a atuação de algumas proteínas das vias na odontogênese e na formação de odontomas e tumores odontogênicos mistos benignos, para isto, foi desenvolvido um estudo seccional restrospectivo e imuno-histoquímico contendo 23 odontomas compostos, 21 odontomas complexos, 17 germes dentários, 05 fibro-odontomas ameloblásticos e 01 fibroma ameloblástico. Os resultados encontrados demonstraram maiores imunoexpressões da via WNT/ß-catenina no epitélio dos germes dentários (p<0,001) e no fibroma ameloblástico, enquanto que, esteve no ectomesênquima dos odontomas (p<0,001) e fibro-odontomas ameloblásticos. A via WNT/ßcatenina correlacionou-se moderadamente e significativamente com a CK14 no epitélio (p = 0,007) dos odontomas. A BMP4 foi imunoexpressa, especialmente, no ectomesênquima dos odontomas complexos (mediana = 33,7; p<0,001). A via Shh foi mais imunoexpressa no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p=0,029). De forma similar, o TGFß apresentou maior imunoexpressão no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p = 0,002). O dente em desenvolvimento exibiu maiores concentrações para estas proteínas no epitélio odontogênico nas fases de botão e capuz e a expressão diferencial ocorreu, principalmente, no ectomesênquima dos tumores, o que indica que esse componente é de fato mais proliferativo (AU).

Tooth development depends on a series of reciprocal signaling interactions between oral epithelium (EO) and neural crest-derived ectomesenchyme, the WNT pathway with TGF-ß and BMP4 has been implicated in tumorigenesis. The Wingless (Wnt)/ß-catenin signaling pathway is essential for the early activation of odontogenesis and the development of odontogenic tumors. TGF-ß and BMPs have been associated with reactionary and reparative dentinogenesis processes. Shh signaling can regulate cell proliferation in dental ectomesenchyme, thus controlling dental morphogenesis. The objective of the research was to investigate the role of some proteins in the pathways in odontogenesis and in the formation of odontomas and benign mixed odontogenic tumors. tooth germs, 05 ameloblastic fibro-odontomas and 01 ameloblastic fibroma. The results found showed higher immunoexpressions of the WNT/ß-catenin pathway in the epithelium of tooth germs (p<0.001) and in ameloblastic fibroma, while it was in the ectomesenchyme of odontomas (p<0.001) and ameloblastic fibroodontomas. The WNT/ß-catenin pathway correlated moderately and significantly with CK14 in the epithelium (p = 0.007) of odontomas. BMP4 was immunoexpressed, especially in the ectomesenchyme of complex odontomas (median = 33.7; p<0.001). The Shh pathway was more immunoexpressed in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p=0.029). Similarly, TGF-ß showed higher immunoexpression in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p = 0.002). The developing tooth exhibited higher concentrations of these proteins in the odontogenic epithelium in the bud and cap phases and the differential expression occurred mainly in the ectomesenchyme of the tumors, which indicates that this component is in fact more proliferative (AU).

The role of nuclear transcription factor Gli in the regulation of hepatic epithelial mesenchymal transition in biliary atresia mice caused by Rhesus monkey rotavirus infection / 中华实用儿科临床杂志

Objective:To investigate the role of nuclear transcription factor Gli1/Gli2 of the sonic hedgehog (Shh) signaling pathway in the hepatic epithelial mesenchymal transition (EMT) of biliary atresia mice caused by Rhesus rotavirus (RRV) infection.Methods:The biliary atresia model in mice was generated by RRV infection.Mice were divided into normal group, model group, Gli1 overexpression group, Gli1 shRNA group, Gli2 overexpression group and Gli2 shRNA group.Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expressions of regulatory factors for EMT (Snail/Slug) and characteristic cytokines of EMT [Vimentin, α-smooth muscle actin(α-SMA), E-cadherin] in mouse liver tissues.Additionally, hematoxylin-eosin staining and Masson staining were performed to calculate the percentage of liver fibrous tissue expression area.The data were analyzed by One- Way ANOVA and LSD- t test. Results:The relative mRNA expression of Snail, Slug, Vimentin, α-SMA and E-cadherin in Gli2 overexpression group, Gli2 shRNA group and model group were 15.13±3.40, 5.48±0.46, 8.78±1.06, 12.40±2.18 and 3.06±0.53; 3.73±1.16, 5.62±1.75, 3.56±1.06, 3.88±1.16 and 10.51±1.83; 8.13±1.27, 5.32±0.98, 5.05±0.98, 4.02±0.77 and 5.12±1.60.Compared with those of the model group, mRNA levels of Snail, Vimentin and α-SMA were significantly higher in Gli2 overexpression group, while that of E-cadherin was significantly lower( t=4.53, 5.29, 8.12, -2.13; all P<0.05); compared with those of the model group, mRNA levels of Snail and Vimentin in Gli2 shRNA group significantly decreased, while that of E-cadherin significantly increased( t=-2.86, -2.12, 5.62; all P<0.05). In Gli2 overexpression group, Gli2 shRNA group and model group, the protein levels of Snail, Slug, Vimentin, α-SMA and E-cadherin were 2.02±0.39, 0.31±0.08, 0.95±0.17, 1.07±0.17 and 0.42±0.06; 0.53±0.13, 0.40±0.18, 0.20±0.04, 0.28±0.07 and 1.09±0.31; 0.70±0.15, 0.42±0.22, 0.64±0.13, 0.81±0.11 and 0.42±0.09.Compared with those of the model group, protein levels of Snail, Vimentin and α-SMA were significantly higher in Gli2 overexpression group( t=12.71, 4.28, 3.70; all P<0.05); compared with those of the model group, protein levels of Vimentin and α-SMA in Gli2 shRNA group significantly decreased, while that of E-cadherin significantly increased( t=-6.14, -7.57, 5.96; all P<0.05). However, no significant change trend were detected in expression levels of characteristic cytokines of EMT between Gli1 overexpression group and Gli1 shRNA group.The area percentage of liver fiber expression in normal group, model group, Gli1 overexpression group, Gli1 shRNA group, Gli2 overexpression group and Gli2 shRNA group were (1.03±0.58)%, (33.02±11.39)%, (39.81±5.67)%, (26.06±1.29)%, (49.81±8.57)% and (17.55±0.66)%, respectively.Besides, in terms of percentage of area expressed in liver fiber tissue, the Gli2 overexpression group and Gli2 shRNA group were statistically significant compared with the model group( t=3.21, -2.96; all P<0.05), while the Gli1 overexpression group and Gli1 shRNA group were not statistically significant compared with the model group (all P>0.05). Conclusions:The Shh signaling pathway plays an important role in liver fibrosis in mice with biliary atresia.Gli2, a key transcription factor of Shh signaling pathway, can significantly regulate liver EMT process in mice with biliary atresi.

Role of Shh/Gli1 signaling pathway in sleep deprivation-induced cognitive impairment in young mice / 中华麻醉学杂志

Objective:To evaluate the role of sonic hedgehog (Shh)/glioma-associated oncogene homolog 1 (Gli1) signaling pathway in sleep deprivation-induced cognitive impairment in young mice.Methods:Forty-eight SPF healthy male C57BL/6 mice, aged 4 weeks, weighing 14-16 g, were divided into 3 groups ( n=16 each) by the random number table method: control group (C group), sleep deprivation group (SD group) and Shh agonist SAG group (SD+ SAG group). Multi-platform water environment method was used to prepare the sleep deprivation model in mice, and the sleep deprivation was 20 h a day for 10 consecutive days.In SD+ SAG group, SAG 10 mg/kg was intraperitoneally injected at 5 min before each sleep deprivation, while the equal volume of normal saline was intraperitoneally injected in group C and group SD.The mice underwent novel object recognition and Y-maze tests at 24 h after development of the model.Mice were sacrificed after the behavioral testing, and the hippocampi were isolated for determination of the density of dendritic spines in hippocampal CA1 region (by Golgi staining), expression of Gli1 and brain-derived neurotrophic factor (BDNF) in hippocampal tissues (by Western blot), and expression of Gli1 and BDNF mRNA in hippocampal tissues (by quantitative real-time polymerase chain reaction). Results:Compared with group C, the preference index in novel object recognition and Y-maze tests and density of dendritic spines in CA1 region were significantly decreased, and the expression of Gli1 and BDNF protein and mRNA in hippocampus was down-regulated in group SD ( P<0.05). Compared with group SD, the preference index in novel object recognition and Y-maze tests and density of dendritic spines in CA1 region were significantly increased, and the expression of Gli1 and BDNF protein and mRNA in hippocampus was up-regulated in group SD+ SAG ( P<0.05). Conclusions:Inhibition of Shh/Gli1 signaling pathway and reduction of plasticity of dendritic spines of hippocampal neurons are involved in sleep deprivation-induced cognitive impairment in young mice.

Potential mechanism of NVP-LDE225 combined with radiotherapy for melanoma / 中华放射肿瘤学杂志

Objective:To explore the potential mechanism and feasibility of sonic hedgehog (SHH) signal pathway inhibitor NVP-LDE225 combined with radiotherapy in the treatment of melanoma.Methods:The Gli1 mRNA expression of the melanoma cells (Melan-A and SK-MEL-2) was detected by qPCR. After treatment with NVP-LDE225, GDC-0449 or combined with radiation for 24 hours, the number and activity of Melan-A and SK-MEL-2 were detected by cell counting and CCK-8 kit. The survival and proliferation ratio of Melan-A and SK-MEL-2 were detected by cell cloning. The changes of cell cycle of melanoma Melan-A cells were determined by flow cytometry. The levels of the Bax and Caspase3 of Melan-A apoptosis protein in melanoma cells were detected by Western blot.Results:NVP-LDE225, an inhibitor of Smo, decreased the mRNA expression of Gli1 in the melanoma cells in a dose-dependent manner, inhibited the proliferation ratio of the melanoma cells, induced apoptosis, and arrested melanoma cells in G 0 / G 1 phase. Gamma ray irradiation after NVP-LDE225 treatment further inhibited the SHH signal pathway, arrested the melanoma cells in G 2 / M phase, and further increased the inhibitory effect on melanoma cell proliferation. Conclusions:NVP-LDE225, an inhibitor of Smo, can inhibit SHH signal pathway in melanocytes, suppress cell proliferation, and further increases the effect of inhibiting cell proliferation after combining with irradiation. It can be used as a potential drug in combination with radiotherapy in the treatment of melanoma, which is worthy of further study.

Effects of SET7 on angiotensin II-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.@*METHODS@#Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1).@*RESULTS@#Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all @*CONCLUSIONS@#Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.

Lycium barbarum polysaccharide inhibits the proliferation and promots the apoptosis of osteosarcoma HOS cells by blocking Shh signaling pathway / 中国临床药理学与治疗学

AIM: To study the effects and mechanism of Lycium barbarum polysaccharide on the proliferation and apoptosis of osteosarcoma HOS cells. METHODS: Osteosarcoma HOS cells were divided into four groups: control group, LBP-I group, LBP-II group and LBP-III group. MTT was used to detect cell proliferation; PI single staining was used to detect cell cycle; Annexin V-FITC/PI double staining was used to detect apoptosis; Western blot was used to detect the expression of Cleaved Caspase-3, Cleaved PARP, cyclin-dependent kinase 4 (CDK4), cyclin D1, Shh and Gli1. Shh signal activator and Lycium barbarum polysaccharide treated osteosarcoma HOS cells together; the changes of cell proliferation, cell cycle and apoptosis were observed.RESULTS: Compared with the control group, the proliferation ability of LBP-I group, LBP-II group and LBP-III group decreased, the proportion of cells in G0/G1 phase increased[(51.2±4.1)% vs. (59.1±3.2)%, (66.8±2.0)%, (72.3±3.2)%, F=72.76, P<0.001], the level of apoptosis increased[(3.9±0.3)% vs. (13.2±1.2)%, (17.6±1.3)%, (24.8±2.1)%, F=364.50, P<0.001], the expression levels of Cleaved Caspase-3, Cleaved PARP protein increased, the expression levels of CDK4, cyclin D1, Shh and Gli1 decreased (P<0.05). Compared with cells not treated with Shh signal activator, the cells treated with Shh signal activator could reverse the effect of Lycium barbarum polysaccharide on the proliferation, cycle arrest and apoptosis of osteosarcoma HOS cells. CONCLUSION: Lycium barbarum polysaccharides blocked the cell cycle of osteosarcoma HOS cells, inhibited cell proliferation and promoted cell apoptosis by inhibiting Shh signaling pathway.

Sonic hedgehog-polydopamine-fibrin scaffold promotes recovery of spinal cord injury in rats / 中国组织工程研究

BACKGROUND: Recently, most studies have combined tissue engineering materials with stem cells or factors to improve the microenvironment of animal models of spinal cord injury to increase the duration of action, improve the recovery effect and prognosis. OBJECTIVE: To investigate the effect of sonic hedgehog-polydopamine-fibrin scaffold on the repair of spinal cord injury in rats. METHODS: Fibrin glue was made using a vacuum freeze-dryer. The prepared fibrin glue was immersed in a dopamine hydrochloride solution for 24 hours for cross-linking. Then the cross-linked scaffold was placed in a factor solution for adsorption and cross-linking for 24 hours. Sonic hedgehog-polydopamine-fibrin scaffolds were prepared. Sixty female SD rat models of spinal cord injury were established and then divided into four groups: In the group A, no material was implanted. In the groups B, C and D, fibrin scaffolds, polydopamine-fibrin scaffolds, and sonic hedgehog-polydopamine-fibrin scaffolds were implanted respectively. The Basso, Beattie and Bresnahan (BBB) locomotor scale score of lower limb locomotor function was evaluated within 12 weeks after surgery. At 12 weeks post-surgery, the tissue at the site of spinal cord injury was collected for histological observation (hematoxylin-eosin and immunohistochemical staining) and western blot assay. This study was approved by the Animal Ethics Committee of Jiangsu University, China. RESULTS AND CONCLUSION: (1) From 2 weeks after surgery, the lower limb locomotor function of rats in each group began to recovery. At 5-12 weeks after surgery, the BBB score of group D was significantly higher than that of the other three groups (P < 0.05). Rats in group D had the best recovery of locomotor function of the lower limb. (2) Hematoxylin-eosin staining revealed newly generated nerve fibers in the groups C and D, and that the number of density of new nerve fibers in group C was lower than that in group D. (3) Immunohistochemical staining showed that a large amount of linearly arranged new nerve fibers were observed in the completely transected site of rat spinal cord. In group D, myelin basic protein-, growth related protein- and neurofilament protein-positive rates were significantly higher (P < 0.05), and glial fibrillary acidic protein-positive rate was significantly lower, compared with the other three groups. (4) Western blot assay revealed that in group D, the protein expression of myelin basic protein, growth related protein and neurofilament protein was significantly higher (P < 0.05), and the protein expression of glial fibrillary acidic protein was significantly lower (P < 0.05), compared with the other three groups. (5) These results suggest that sonic hedgehog-polydopamine-fibrin has a good sustained-release performance, which can greatly promote the repair of spinal cord injury in rats.

Desarrollo embriofetal del iris: inmunotinción del morfógeno Shh / Embryo-fetal development of iris: immunohistochemestry of morphogen Shh

RESUMEN: Para que se desarrolle el iris, se requiere una especificación de la capa periférica de la copa óptica a un destino no neuronal y además la migración de células mesenquimales perioculares. Nuestro objetivo fue reconocer los cambios histológicos de los derivados periféricos de la copa óptica y mesénquima periocular, como también reconocer la presencia del morfógeno Sonic hedgehog (Shh) en las capas que constituyen el esbozo de iris. Se utilizaron 15 ratones hembras (Mus musculus) adultas jóvenes gestantes. Se realizó eutanasia con tiopental sódico. Los embriones y fetos de 12, 14,5 y 17 días post-coital (dpc) fueron procesados con técnica histológica e inmunohistoquímica con anticuerpo anti-Shh (scbt, H-160, conejo) con dilución 1:100 en PBS. A los 12 dpc, se observa una cópa óptica que presenta capas retinianas interna y externa, y el iris no se observa. Entre el cristalino y el ectodermo superficial se identifican 4 capas de células mesenquimales. A los 14,5 dpc, el iris contiene dos capas epiteliales (interna y externa) que se continúan con las capas neural y pigmentaria de la retina. Se observan 8 capas de células mesenquimales. A los 17 dpc, la capa epitelial interna del iris presenta un segmento más elongado con inmunotinción positiva a Shh y otra parte que constituye un epitelio de células cilíndricas simples negativas a este anticuerpo. La capa epitelial externa presenta el mismo epitelio inmunonegativo. Las capas de la retina también son positivas, como también la periferia del cristalino. No esta formado el iris ni tampoco el cuerpo ciliar. La inmunopositividad en el cristalino, en el primer segmento de la capa interna del esbozo del iris y en la capa ganglionar retinal a los 17 dpc, se relaciona con la diferenciación tardía del iris y con los ojos cerrados de las crías al nacimiento.

SUMMARY: In order for the iris to develop, a specification of the peripheral layer of the optic cup to a non-neuronal target is required, as well as the migration of periocular mesenchymal cells. Our aim was to recognize the histological changes of peripheral derivatives of the optic cup and periocular mesenchyme, as well as recognize the presence of the morphogen Sonic hedgehog (Shh) in the layers constituting the outline of the iris. 15 female mice (Mus musculus) pregnant young adults were used. Euthanasia was performed with sodium thiopental. Embryos and fetuses of 12, 14.5 and 17 days post-coital (dpc) were processed with histological and immunohistochemical technique with anti-Shh antibody (scbt, H 160, rabbit) with dilution 1:100 in PBS. At 12 dpc, an optic cup showing internal and external retinal layers is observed, and the iris is not observed. Between the lens and the superficial ectoderm, 4 layers of mesenchymal cells are identified. At 14.5 dpc, the iris contains two epithelial layers (internal and external) that are continued with the neural and pigmentary layers of the retina. 8 layers of mesenchymal cells are observed. At 17 dpc, the inner epithelial layer of the iris presents a more elongated segment with positive immunostaining to Shh and another part that constitutes an epithelium of simple cylindrical cells negative to this antibody. The outer epithelial layer presents the same immunonegative epithelium. The layers of the retina are also positive, as well as the periphery of the lens. The iris is not formed nor is the ciliary body.The immunopositivity in the lens, in the first segment of the inner layer of the iris outline and in the retinal ganglion layer at 17 dpc, is related to the late differentiation of the iris and the closed eyes of the offspring at birth.

Inmunolocalización de Sonic hedgehog en el desarrollo embrio-fetal de ratones ( Mus musculus ) / Immunolocalization of Sonic hedgehog in embryo-fetal mouse ( Mus musculus ) development

Sonic hedgehog (Shh) es un morfógeno esencial para el desarrollo de diversas estructuras, tales como notocorda, placa del piso del tubo neural, miembros, entre otros. Se buscó determinar la inmunolocalización de Shh en embriones y fetos de ratón. Para ello, se eutanasiaron 10 ratones gestantes (Mus musculus) BALB/c, un grupo de 5 animales a los 12,5 días post-coito (dpc), y otro grupo a los 17,5 dpc. Los embriones y fetos obtenidos fueron fijados en formalina al 10 % tamponada en PBS e incluidos en paraplast. Se realizaron cortes transversales seriados. Se utilizó anticuerpo policlonal Shh (Santa Cruz Biotechnology, H-160, conejo), dilución 1:100. Se identificó y describió la inmunolocalización de las muestras marcadas positivamente. La expresión de Shh en los embriones de 12,5 dpc fue inmunopositiva en notocorda, placa del piso del tubo neural, precartílago de radio y ulna, y prácticamente todos los epitelios: bronquial, intestinal, vejiga y uretra. En la etapa fetal, a los 17,5 dpc la inmunopositividad desaparece en el cartílago a excepción de zonas de osificación, disminuye en la epidermis pero aparece en folículos pilosos. La mucosa intestinal se ha diferenciado en segmentos, mostrando una inmunotinción mayor a nivel de las vellosidades intestinales. Shh actúa en distintos estadios del periodo gestacional, siendo clave en la diferenciación de distintas estructuras. En etapas embrionaria, es vital en la formación del sistema nervioso, organogénesis y formación de miembros, por lo que su expresión se encuentra en estas zonas. Sin embargo, en la etapa fetal la expresión cambia a estructuras de mayor especialización como folículo piloso y vellosidades intestinales.

Sonic hedgehog (Shh) is an essential morphogen for the development of various structures, such as notochord, neural tube floor plate, limbs, among others. We sought to determine the immunolocalization of Shh in embryos and mouse fetuses. To do this, 10 pregnant mice (Mus musculus) BALB /c were euthanized, a group of 5 animals at 12.5 days postcoitus (dpc), and another group at 17.5 dpc. Embryos and fetuses obtained were fixed in 10 % formalin buffered in PBS and embedded in paraplast. Serial cross sections were made. Polyclonal antibody Shh (Santa Cruz Biotechnology, H-160, rabbit), dilution 1:100 was used. The immunolocalization of the positively labeled samples was identified and described. Shh expression in 12.5 dpc embryos was immunopositive in notochord, neural tube floor plate, radius precartilage and ulna, and practically all epithelia: bronchial, intestinal, bladder and urethra. In the fetal stage, at 17.5 dpc the immunopositivity disappears in the cartilage except for areas of ossification, decreases in the epidermis but appears in hair follicles. The intestinal mucosa has differentiated into segments, showing greater immunostaining at the level of the intestinal villi. Shh acts in different stages of the gestational period, being key in the differentiation of different structures. In embryonic stages, it is vital in the formation of the nervous system, organogenesis and formation of limbs, so its expression is found in these areas. However, in the fetal stage the expression changes to more specialized structures such as hair follicles and intestinal villi.

Roles of Sonic Hedgehog Signaling During Tooth Root and Periodontium Formation / 대한소아치과학회지

The aim of this study was to understand the roles of Sonic Hedgehog (SHH) signaling during tooth root and periodontium formation. In this study, we generated the dental mesenchyme-specific Smoothened (Smo) activated/inactivated mice with the activity of Cre recombinase under the control of osteocalcin promoter.In the Smo activated mutant molar sections at the postnatal 28 days, we found extremely thin root dentin and widened pulp chamber. Picrosirius red staining showed loosely arranged fibers in the periodontal space and decreased cellular cementum with some root resorption. Immunohistochemical staining showed less localization of matrix proteins such as Bsp, Dmp1, Pstn, and Ank in the cementum, periodontal ligament, and/or cementoblast.In the Smo inactivated mutant mouse, there was not any remarkable differences in the localization of these matrix proteins compared with the wild type. These findings suggest that adequate suppressing regulation of SHH signaling is required in the development of tooth root and periodontium.

Advances in Shh signaling pathway of renal interstitial fibrosis / 国际儿科学杂志

Sonic hedgehog signaling pathway has a unique role in the process of renal fibrosis,while it is mainly expressed in the impaired renal tubular epithelial cells. Shh signaling pathway promotes the process of epithelial-mesenchymal transition and renal tubulointerstitial fibrosis. Shh signaling pathway ligand triggers cell proliferation in the pericyte,suggesting a possible role for this pathway in the regulation of cell cycle progression of myofibroblasts progenitors during the development of renal fibrosis. Shh protein can be expressed at the early stage of kidney injury,which indicates that Shh signaling pathway is involved in the tissue repair process after kidney injury at the early stage. The process of renal interstitial fibrosis related to signaling pathway is involved with signaling pathway and proteins,cytokines,extracellular matrix,complement and so on. This paper reviews the progress in the study of the relationship between Shh and renal interstitial fibrosis.

Progress of targeting inhibition of Sonic Hedgehog signaling pathway in leukemia / 白血病·淋巴瘤

Sonic Hedgehog (SHH) signaling pathway participates in the regulation of various organs and growth of tissue cells, maintains normal function and structure in the development of embryogenesis, however, SHH signaling pathway is in a state of inhibition. Meanwhile, the abnormal regulation of SHH signaling pathway plays an important role in the occurrence, development and drug resistance of leukemia. The mechanism of SHH signaling pathway in leukemia is similar to the solid tumor. With the further understanding of SHH signaling pathway, there are many antitumor drugs targeting SHH signaling pathway. The targeting inhibitors targeted SHH signaling pathway will become a new method for the treatment of leukemia. This paper reviews the application of the inhibitors targeting SHH signaling pathway in leukemia.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts DNA protection against hypoxia / 中国药理学通报

Aim To investigate the protective roles of sonic hedgehog( Shh) signaling pathway in hypoxia-in-duced DNA damage with the neonatal rat cardiomyo-cytes. Methods The hypoxia model on neonatal car-diomyocytes was established with one to two days old Sprague Dawley rats by deprivation of oxygen and glu-cose ( OGD) . After pretreated with Shh pathway ago-nist SAG1.3 or antagonist GANT61, the survival rates of cardiomyocytes were assayed by MTT after OGD 6 hours or 12 hours. The protein levels of Shh pathway, phosphorylated histone H2AX at serine 139 (γH2AX), phosphorylated ATM (p-ATM), phospho-rylated p53 ( p-p53 ) , cleaved-caspase-3, Bcl-2 and Bax were detected by Western blot. The γH2AX foci was detected by immunofluorescence. Results Com-pared to control group, the protein expression of γH2AX, p-ATM, cleaved-caspase-3, p-p53 in OGD cardiomyocytes significantly increased, and Bcl-2/Bax ratio proportionally decreased. Particularly, the ex-pression of γH2AX, p-ATM was highest at OGD 6 h, and then gradually declined after OGD 12 h. After SAG1.3 pretreatment, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 dramatically de-creased and the Bcl2/Bax ratio increased in OGD 6 h or OGD 12 h cardiomyocytes. On the contrary, in GANT61 pretreatment group, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 signifi-cantly increased and the Bcl-2/Bax ratio decreased compared to the OGD 6 h or OGD 12 h cardiomyo-cytes. Conclusion The activation of Shh pathway protects cardiomyocytes against hypoxia-induced apop-tosis through inhibition of DNA damage.

The study on the relationship between the levels of sonic hedgehog and vascular endothelial growth factor in serum and cerebral collateral circulation in patients with symptomatic cerebral artery stenosis / 中国医师进修杂志

Objective To investigate the changes of the levels of sonic hedgehog (SHH) and vascular endothelial growth factor (VEGF) in serum and its relationship with collateral circulation in patients with symptomatic middle cerebral artery stenosis. Methods From January 2015 to January 2018, a total of 268 patients with acute ischemic stroke confirmed as unilateral middle cerebral artery M1 segment (MCA-M1) severe stenosis or occlusion by digital subtract angiography (DSA) were enrolled. The baseline clinical data were collected. According to the establishment of collateral circulation shown by DSA, they were divided into good collateral circulation group (152 patients) and poor collateral circulation group (116 patients). The levels of SHH and VEGF in serum were detected by enzyme linked immunosorbent assay (ELISA), the expression characteristics of SHH and VEGF in serum and the relative factors influencing the establishment of collateral circulation were analyzed. Results The levels of serum SHH and VEGF in good collateral circulation group were significantly higher than those in poor collateral circulation group (P < 0.01). Pearson correlation analysis showed that there was a positive correlation between SHH and VEGF (r=0.758, P < 0.01). Multivariate Logistic regression analysis showed that the levels of serum SHH ( OR=0.310, 95% CI 0.117-0.819, P=0.018) and VEGF ( OR=0.361, 95% CI 0.147-0.887, P=0.026) were independent protective factors for the establishment of collateral circulation. Diabetes ( OR=3.094, 95% CI 1.321-7.245, P=0.009) was independent risk factor for the establishment of collateral circulation. Conclusions The levels of serum SHH and VEGF are closely related to the formation of collateral circulation and they are independent protective factors. SHH may be involved in the establishment of cerebral collateral circulation by regulating the expression of VEGF and diabetes is not conducive to the formation of collateral circulation.

The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide / 中华急诊医学杂志

Objective To explore the effect of expression of protein kinase C receptor 1(RACK1) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC).Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Anhui province.Using immunocytochemistry method,the expression of RACK1 protein in RPMVECs was detected,cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group,SAG(smoothened Agonist,a SHH signaling pathway specific agonist) dose-dependent group,LPS time-dependent group,SAG time-dependent group and LPS+SAG group.In LPS dose-dependent groups,RPMVECs were cultured with 0.1,1,10 mg/L LPS for 8 h.In LPS time-dependent groups,RPMVECs were cultured with 10 mg/L LPS for 0,2,4,8,12,24 h.In SAG dose-dependent groups,RPMVECs were cultured with 0.1,1,10 μ mol/L for 8 h.In SAG time-dependent groups,RPMVECs were cultured with 1 μ mol/L SAG for 0,2,4,8,12,24 h.In LPS+SAG group,RPMVECs were cultured with 1 μ mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h.In addition,blank group,LPS group and SAG group were set for control.Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention.Results Immunocytochemistry revealed that RACK1 were present in RPMVEC.1.In LPS dose-dependent groups (0,0.1,1,10 mg/L),the level of RACK1 elevated as LPS dose increased correspondingly with inter-group difference (P＜0.05);the relative expression levels of GLI-1 mRNA were (1.109 + 0.063),(1.039 + 0.135),(0.813 ± 0.066),(0.770 + 0.105),(1 mg/L vs.10 mg/L,P＞0.05;the rest P＜0.05).In LPS time-dependent groups,the relative expression level of RACK1 at 2 h (0.370 + 0.010) was higher than that at 0 h (0.329 ± 0.008),peaked at 12 h (1.296 ± 0.048),and compared with 0 h,there was significant differences (F=1 272.204,P＜0.05).The relative expression level of GLI-1 mRNA was decreased at 2 h (0.929 ±-0.007),and compared with 0 h(1.089 ± 0.042),there was significant differences (F=306.609,P＜0.05).2.In SAG dose-dependent groups,there was no significant difference in level of RACK1 between groups(all P＞0.05).The relative expression levels ofGLI-1 mRNA were (1.109 ± 0.063),(1.169 ± 0.052),(3.468 ± 0.128),(3.434 ± 0.054),(0 μ mol/L vs.0.1 μ mol/L and 1 μ mol/L vs.10 μ mol/L,P＞0.05,the rest P＜0.05).Among SAG time-dependent groups,there was no significant difference in levels of RACK1 protein(P＞0.05).The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065),and compared with 0 h (2.651 + 0.123),there was significant differences (F=132.841,P＜0.05).3.In LPS+SAG intervention groups,the expression of RACKI was lower than that in LPS group (0.831 ±0.040 vs.1.189 ± 0.149,P＜0.05),and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 + 0.130 vs.0.796-4-0.082,P＜0.05).Conclusions The LPS up-regulates the expression of RACK1 in RPMVECs,and the activated SHH signaling pathway can down-regulate the expression of RACK1 induced by LPS in RPMVECs.

Hypospadias induced by maternal exposure to di-n-butyl phthalate and its mechanisms in male rat offspring / 中华男科学杂志

Objective@#To induce hypospadias in male rat offspring by maternal exposure to di-n-butyl phthalate (DBP) during late pregnancy and further investigate its mechanisms.@*METHODS@#We randomly divided 20 pregnant rats into a DBP exposure and a control group, the former treated intragastrically with DBP while the latter with soybean oil at 750 mg per kilogram of the body weight per day from gestation days (GD) 14 to 18. On postnatal day (PND) 1, we recorded the incidence rate of hypospadias and observed the histopathological changes in the genital tubercle of the hypospadiac rats. We also measured the level of serum testosterone (T) by radioimmunoassay and determined the mRNA and protein expressions of the androgen receptor (AR), sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor 8 (Fgf8) in the genital tubercle by real-time PCR and Western blot.@*RESULTS@#No hypospadiac male rats were found in the control group. The incidence rate of hypospadias in male offspring was 43.6% in the DBP-treatment group. Histological analysis confirmed hypospadiac malformation. The serum testosterone concentration was decreased in the hypospadiac male rats as compared with the controls (［0.49 ± 0.05］ vs ［1.12 ± 0.05］ ng/ml, P <0.05). The mRNA expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle were significantly lower in the hypospadiac male rats than in the controls (AR: 0.50 ± 0.05 vs 1.00 ± 0.12, P <0.05; Shh: 0.65 ± 0.07 vs 1.00 ± 0.15, P <0.05; Bmp4: 0.42 ± 0.05 vs 1.00 ± 0.13, P <0.05; Fgf8: 0.46 ± 0.04 vs 1.00 ± 0.12, P <0.05), and so were their protein expressions (AR: 0.34 ± 0.05 vs 1.00 ± 0.09, P <0.05; Shh: 0.51 ± 0.07 vs 1.00 ± 0.12, P <0.05; Bmp4: 0.43 ± 0.05 vs 1.00 ± 0.11, P <0.05; Fgf8: 0.57 ± 0.04 vs 1.00 ± 0.13, P <0.05).@*CONCLUSIONS@#Maternal exposure to DBP during late pregnancy can induce hypospadias in the male rat offspring. DBP affects the development of the genital tubercle by reducing the serum T concentration and expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle, which might underlie the mechanism of DBP inducing hypospadias.

Sonic Hedgehog promotes fibroblast-like synoviocytes proliferation via modulating the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway in rheumatoid arthritis / 中华风湿病学杂志

Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

Relationship between Sonic hedgehog signaling pathways and radioresistance of esophageal cancer / 中国病理生理杂志

AIM:To investigate the potential role of Sonic hedgehog (Shh) signaling pathways in the radioresistance of esophageal cancer.METHODS:Radioresistant cell line Eca109R was established by repeating X-ray irradiation at dose of 60 Gy in total using Eca109 cells as parental cells.The radiosensitivity of the parental and radioresistant cells was confirmed by colony formation assay.The cell viability was detected by CCK-8 assay.The intracellular protein levels of Shh and Gli1 were determined by Western blot and immunofluorescence.RESULTS:The survival fractions at dose of 2 Gy for Eca109R cells and Eca109 cells were 0.937±0.013 and 0.499±0.042, respectively.The inhibitory rate of cell viability decreased gradually in the Eca109R cells (P<0.05), suggesting that the radioresistant cell line was successfully established.The results of Western blot indicated that the protein expression of Shh and Gli1 was much higher in the Eca109R cells than that in the Eca109 cells (P<0.05).Immunofluorescence staining showed that Gli1 was expressed in the cytoplasm and nucleus, and presented nuclear clustering in the Eca109R cells.The positive rate of Gil1 expression in Eca109 cells was 52.3%± 0.035%, while that in Eca109R cells was 87.6%±0.021% (P<0.05).CONCLUSION:The radioresistance of esophageal cancer may be related to the activation of Shh signaling pathways with over-expression of Gli1 and other related proteins.

Mechanism of Ezhu-containing serum in inhibiting expression of Shh and Gli1 in hepatic stellate cells / 中国中药杂志

To explore the mechanism of Ezhu-containing serum in inhibiting the expression of sonic hedgehog(Shh) and glioma-associated oncogene homolog-1(Gli1) in hepatic stellate cells(HSCs) induced by leptin. Twenty sprague-dawley (SD) rats were randomly divided into 2 groups (n=10), and given Ezhu-decoction and physiological saline by gavage for 10 days to prepare drug-containing serums. The HSCs during the exponential growth phase were divided into 7 groups： blank control group, model group, hedgehog pathway inhibitor(cyclopamine) group, Ezhu group, Ezhu and cyclopamine group, hedgehog pathway agonost(pumorphamine) group, Ezhu and purmorphamine group. HSCs were cultured in vitro and induced with 100 μg•L ⁻¹ leptin(except for the blank control group), then treated separately with the corresponding drugs for 24 hours. After the cells were collected, HSCs proliferation was detected using MTT colorimetric assay; the expressions of Shh and Gli1 were determined by PT-PCR, Western blot and immunofluorescence, respectively. The expressions of Shh and Gli1 were significantly increased after the HSCs of rats were induced by leptin (compared with the blank control group, P<0.01). After being interfered with Hh pathway inhibitor (cyclopamine) and Ezhu-containing serum, the expressions of Shh and Gli1 were decreased significantly(compared with the model group, P<0.01). After Ezhu-containing serum was used to interfere the Hh pathway inhibitor group, the mRNA and protein expressions of Shh and Gli1 were decreased significantly(compared with the model group, P<0.01). After Ezhu-containing serum was used to interfere the purmorphamine group, the mRNA and protein expressions of Shh and Gli1 decreased significantly(compared with the purmorphamine group, P<0.01). Ezhu-containing serum plays an important role in inhibiting HSCs activation by taking part in hedgehog signaling pathway, so as to regulate the expression of Shh and Gli1 in leptin-induced HSCs and then inhibit liver fibrosis.