Intervention effects and related mechanisms of glycyrrhizic acid on zebrafish with Hirschsprung-associated enterocolitis.

BACKGROUND: The prevention and treatment of Hirschsprung-associated enterocolitis (HAEC) is a serious challenge in pediatric surgery. Exploring the mechanism of HAEC is conducive to the prevention of this disease. AIM: To explore the possible mechanism of glycyrrhizic acid (GA) and its therapeutic effect on HAEC. METHODS: We developed a model of enteritis induced by trinitrobenzenesulfonic acid (TNBS) in zebrafish, and treated it with different concentrations of GA. We analyzed the effect of GA on the phenotype and inflammation of zebrafish. RESULTS: After treatment with TNBS, the area of the intestinal lumen in zebrafish was significantly increased, but the number of goblet cells in the intestinal lumen was significantly reduced, but these did not increase the mortality of zebrafish, indicating that the zebrafish enteritis model was successfully developed. Different concentrations of GA protected zebrafish with enteritis. In particular, high concentrations of GA were important for the prevention and control of HAEC because it significantly reduced the intestinal luminal area, increased the number of goblet cells in the intestinal lumen, and reduced the levels of interleukin (IL)-1ß and IL-8. CONCLUSION: GA significantly reduced the intestinal luminal area, increased the number of intestinal goblet cells, and decreased IL-1ß and IL-8 in zebrafish, and is important for prevention and control of HAEC.

Expression of specificity protein 1 and collagen I in primary pterygial tissues / Expressão da proteína de especificidade 1 e colágeno I em tecidos pterigiais primários

ABSTRACT Purpose: To determine the expression profiles of the transcription factor specificity protein 1 and collagen I in primary pterygial and normal conjunctival tissues, and to explore the role of specificity protein 1 and collagen I in pterygial development. Methods: The pterygial tissues of 20 patients who underwent resection of primary pterygial tissue in our hospital from June 2016 to December 2017 and the conjunctival tissues of 10 patients with enucleation due to trauma were collected. Reverse transcription quantitative-po lymerase chain reaction and western blot analyses were used to detect the relative expression levels of specificity protein 1 and type I collagen at the mRNA and protein levels. Results: The content of specificity protein 1 and collagen I mRNA and protein was significantly greater in primary pterygial tissue than it was in conjunctival tissue (p<0.05). There was a positive correlation between the mRNA and protein levels of specificity protein 1 and collagen I in primary pterygial tissues (protein: r=1, p<0.05; mRNA: r=1, p<0.05). Conclusion: Specificity protein 1 and collagen I are expressed in normal conjunctival and pterygial tissues, but expression is significantly greater in the latter. Specificity protein 1 and collagen I may be involved in the regulation of the development of primary pterygium.

RESUMO Objetivo: Determinar os perfis de expressão do fator de transcrição da proteína de especificidade 1 e do colágeno I em tecidos pterigiais primários e conjuntivais normais, e explorar o papel da proteína de especificidade 1 e colágeno I no desenvolvimento pterigial. Métodos: Foram coletados os tecidos pterigiais de 20 pacientes submetidos à ressecção de tecido de pterígio primário em nosso hospital no período de junho de 2016 a dezembro de 2017 e os tecidos conjuntivais de 10 pacientes com enucleação por trauma. A reação em cadeia da polimerase quantitativa de transcriptase reversa e a análise de Western blot foram utilizadas para detectar os níveis de expressão relativa da proteína de especificidade 1 e colágeno tipo I nos níveis de mRNA e proteína. Resultados: O conteúdo de especificidade da proteína 1 e do mRNA e proteína do colágeno I foi significativamente maior no tecido de pterígio primário do que no tecido conjuntival (p<0,05). Houve correlação positiva entre os níveis de mRNAs e proteína de especificidade 1 e colágeno I nos tecidos primários do pterígio (proteínas: r=1, p<0,05; mRNA: r=1, p<0,05). Conclusão: A proteína de especificidade 1 e do colágeno I é expressa nos tecidos conjuntivais e pterigiais normais, mas a expressão é significativamente maior no segundo. A especificidade da proteína 1 e do colágeno I pode ser envolvida na regulação do desenvolvimento do pterígio primário.

Dynamic changes of activator protein 1 and collagen I expression in the sclera of myopia guinea pigs.

AIM: To investigate the dynamic changes of activator protein 1 (AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6wk (FDM group). Normal control group (n=25) were untreated. Changes in refractive power and axial length (AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6wk, and 4/-1wk of form-deprivation, the diopter in the FDM group was gradually changed (2.08±0.31, -1.23±0.68, -4.17±0.58, -7.07±0.55, and -2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased (5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated (all P<0.05); the mRNA expressions of them were also gradually down-regulated (all P<0.05); and there was positive correlation between them. The control group had no obvious change in each index (all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.

Expression of specificity protein 1 and collagen I in primary pterygial tissues.

PURPOSE: To determine the expression profiles of the transcription factor specificity protein 1 and collagen I in primary pterygial and normal conjunctival tissues, and to explore the role of specificity protein 1 and collagen I in pterygial development. METHODS: The pterygial tissues of 20 patients who underwent resection of primary pterygial tissue in our hospital from June 2016 to December 2017 and the conjunctival tissues of 10 patients with enucleation due to trauma were collected. Reverse transcription quantitative-po lymerase chain reaction and western blot analyses were used to detect the relative expression levels of specificity protein 1 and type I collagen at the mRNA and protein levels. RESULTS: The content of specificity protein 1 and collagen I mRNA and protein was significantly greater in primary pterygial tissue than it was in conjunctival tissue (p<0.05). There was a positive correlation between the mRNA and protein levels of specificity protein 1 and collagen I in primary pterygial tissues (protein: r=1, p<0.05; mRNA: r=1, p<0.05). CONCLUSION: Specificity protein 1 and collagen I are expressed in normal conjunctival and pterygial tissues, but expression is significantly greater in the latter. Specificity protein 1 and collagen I may be involved in the regulation of the development of primary pterygium.

Expression and role of specificity protein 1 in the sclera remodeling of experimental myopia in guinea pigs.

AIM: To study the expression of collagen I and transcription factor specificity protein 1 (Sp1), a transforming growth factor-ß1 (TGF-ß1) downstream target, and reveal the impact of the TGF-ß1-Sp1 signaling pathway on collagen remodeling in myopic sclera. METHODS: Seventy-five 1-week-old guinea pigs were randomly divided into normal control, form deprivation myopia (FDM), and self-control groups. FDM was induced for different times using coverage with translucent latex balloons and FDM recovery was performed for 1wk after 4wk treatment; then, changes in refractive power and axial length were measured. Immunohistochemistry and reverse transcription-polymerase chain reaction were used to evaluate dynamic changes in collagen I and Sp1 expression in the sclera of guinea pigs with emmetropia and experimental myopia, and the relationship between collagen I and Sp1 levels was analyzed. RESULTS: In the FDM group, the refractive power was gradually changed (from 2.09±0.30 D at week 0 to -1.23±0.69 D, -4.17±0.59 D, -7.07±0.56 D, and -4.30±0.58 D at weeks 2, 4, 6, and 1wk after 4wk, respectively; P<0.05), indicating deepening of myopia. The axial length was increased (from 5.92±0.39 mm at week 0 to 6.62±0.36 mm, 7.30±0.34 mm, 7.99±0.32 mm, and 7.41±0.36 mm at weeks 2, 4, 6, and 1wk after 4wk; P<0.05). The mRNA and protein expression of Sp1 and collagen I in the sclera of the FDM group was lower than that of the control groups (P<0.05), and the reduction was eye-coverage time-dependent. Furthermore, correlation between Sp1 and collagen I down-regulation in the myopic sclera was observed. CONCLUSION: Our data indicate that transcription factor Sp1 may be involved in the regulation of type I collagen synthesis/degradation during myopic sclera remodeling, suggesting that TGF-ß1 signaling plays a role in the development and progression of myopia.

Effects of scleral encircling surgery on vitreous cavity length and diopter.

AIM: To observe the changes of vitreous cavity length and diopter after scleral encircling (SE) produce. METHODS: This prospective study included 68 eyes of 68 non-consecutive patients with macula-off retinal detachment who were operated by SE surgery. The corneal refractive power, ocular axial length and diopter were measured by keratometer, A-mode ultrasonic meter and computed dioptometer. RESULTS: There was no significant difference in corneal refractive power among preoperative and postoperative 1, 3 and 6 mo (0.57±0.54 D at pre-surgery;0.72±0.26 D at 1mo; 0.71±0.34 D at 3mo; 0.69±0.31 D at 6mo; all P>0.05 ). Axial lengths were obviously lengthened, especially in vitreous cavity length (17.87±3.09 mm, 19.69±3.12 mm, 18.97±3.56 mm, 18.76±3.47 mm, 18.68±3.42 mm at pre-surgery, 1wk, 1, 3 and 6mo postoperatively, P <0.05) and diopter also increased at beginning and then recovered gradually. After 1 and 3 mo, axial length (vitreous cavity length) and myopia were more and in higher degree than before surgery. CONCLUSION: The change of postoperative vitreous cavity length is the main factor that results in the changes of axial length and then makes the change of diopter.

Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment.

AIM: To investigate whether photoreceptor necroptosis induced by z-VAD-FMK (pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment. METHODS: Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1. Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8 antibody. RESULTS: It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase (RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptor necroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction. CONCLUSION: These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

[Effects of blocking activation of IGF-1-Stat3 signaling pathway in guinea pig sclera fibroblast by AG490 on expression of MMP-2 and Integrinß(1)].

OBJECTIVE: To explore the effect of Blocking activation of IGF-1-Stat3 signaling pathway in guinea pig sclera fibroblast (GSFs) by AG490 on expressions of MMP-2, Integrinß(1). METHODS: Cultured GSFs were divided into four groups: group A(control group: only DMEM without IGF-1), group B (only IGF-1 group), group C(IGF-1 + PBS group), group D (IGF-1 + 25 µmol/L AG490 group). The expressions of Stat3, p-Stat3, MMP-2, Integrinß(1) protein induced by IGF-1 and inhibited by AG490 in GSFs were detected by Western blot. The levels of Stat3, MMP-2 and Integrinß(1)mRNA were detected by RT-PCR. RESULTS: Compared with Groups A and D, Stat3, p-Stat3, MMP-2 protein expression in groups B and C were expressed at higher level (t(pr) = -32.324, -26.284, -32.876, -26.345, -68.668, -58.724, -187.481, -58.842, -110.264, -120.256, -121.345, -120.286; t(mRNA) = -31.554, -31.178, -31.286, -31.198, -12.076, -14.969, -11.896, -14.546, P < 0.05), but the expression levels were not obviously different between groups B and C (t(p) = -32.720, -32.816, -68.668, -187.481, -110.264, -121.345; t(mRNA) = -0.692, -0.579, P > 0.05), which were similar to mRNA level. The Integrinß(1) protein and mRNA were expressed in groups A, B, C and D but no significant difference among them respectively (F(pr) = 0.214;F(mRNA) = 0.045, P > 0.05). CONCLUSIONS: Activation of Stat3 signaling pathway may be involved in up-modulating the expression of MMP-2 in GSFs, and not affect the Integrinß(1) protein and mRNA changes. The results reveal that Stat3 signaling transduction pathway may play a critical role in sclera remodeling by means of modulating MMP-2 expression.

[Insulin-like growth factor-1 induced activation and expression of signal transducers and activators of transcription-3 in scleral fibroblast of guinea pigs].

OBJECTIVE: To investigate the expression of signal transducers and activators of transcription 3 (STAT3) in sclera fibroblast of guinea pigs for understanding whether STAT3 signaling transduction pathway induced by the insulin-like growth factor-1 (IGF-1) was constitutively activated. METHODS: Immunocytochemical staining was used to determine the protein levels of STAT3 and P-STAT3 (activated STAT3) in scleral fibroblasts. RT-PCR was used to detect the mRNA of STAT3. The effects of IGF-1 on scleral fibroblast proliferation were measured by MTT assay. RESULTS: Immunocytochemical staining and RT-PCR revealed that STAT3 protein and mRNA were over-expressed in the cells which contained constitutively activated STAT3 signaling transduction pathway (t=-6.925, -10.179 and 9.363; P<0.001). The results of MTT showed that IGF-1 induced fibroblasts proliferation significantly at concentrations of 0.5, 1.0, 10.0 microg/L (P<0.05). CONCLUSION: STAT3 signaling transduction pathway is constitutively activated in the treated scleral cells by IGF-1, suggesting that STAT3 signaling transduction pathway may play a critical role in the occurrence of myopia and sclera remodeling.