Imunoexpressão de clic4, α-sma, e-caderina e vimentina em lesões odontogênicas epitelias benígnas / Immunoexpression of clic4, α-sma, e-cadherin and vimentin in benign epithelial odontogenic lesions

As lesões odontogênicas epiteliais benignas constituem um grupo heterogêneo de lesões. A proteína CLIC4 atua na regulação dos processos de parada de crescimento e apoptose, participando também do processo de transdiferenciação dos fibroblastos em miofibroblastos que passam a expressar α-SMA. Além disso, a expressão de CLIC4 pode interferir no processo de transição epitélio-mesenquima (TEM) em neoplasias. Este trabalho avaliou a imunoexpressão de CLIC4, α-SMA, E-caderina e Vimentina em ameloblastomas (AM) (n = 16), ceratocistos odontogênicos (n = 20) e tumores odontogênicos adenomatóides (TOA) (n = 8). A análise da expressão imunoistoquímica das proteínas CLIC4, E-caderina e vimentina no componente epitelial das lesões e de CLIC4 e α-SMA no tecido conjuntivo foi realizada de forma semi-quantitativa por um avaliador previamente calibrado. A expressão no componente epitelial de CLIC4 foi analisada separadamente no núcleo e no citoplasma, bem como a marcação de E-caderina que foi avaliada na membrana e no citoplasma. As comparações dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de CLIC4, α-SMA, E-caderina e Vimentina foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foram observados diferentes padrões de marcação entre os grupos analisados, observando-se que a imunoexpressão exclusivamente citoplasmática da CLIC4 no componente epitelial dos AM (p < 0,001) e TOA (p < 0,001) foi significativamente superior a dos CO, não demonstrarando significância estatística entre os AM e TOA. A imunoexpressão (nuclear e citoplasmática) da CLIC4 no revestimento epitelial CO foi significativamente superior à encontrada no componente epitelial dos AM (p < 0,001) e dos TOA (p < 0,001). A imunoexpressão estromal de CLIC4 foi significativamente superior nos AM (p = 0,009) e CO (p = 0,004) quando comparados aos TOA. A imunoexpressao de α-SMA significativamente maior em AM (p = 0,016) e CO (p = 0,034) quando comparados aos TOA. Para a imunoexpressão membranar da E-caderina em CO foi significativamente superior em comparação à encontrada nos AM (p = 0,009) e nos TOA (p = 0,024). Foi observada maior imunoexpressão de E-caderina (membranar e citoplasmática) nos COs, quando comparados aos AM (p < 0,001) e aos TOAs (p < 0,001). A expressão de Ecaderina citoplasmática foi significativamente maior nos AM e TOA (p < 0,001) quando comparados aos CO. Observou-se diferença estatisticamente significativa na imunoexpressão de vimentina entre os casos de AM e os casos de TOA (p = 0,038) e CO (p < 0,001), bem como entre o TOA e CO (p < 0,001). As correlações testadas entre os escores das proteínas estudadas evidenciou que no grupo dos AM foi possível evidenciar moderada correlação positiva e estatisticamente significativa (r = 0,527; p = 0,036) entre a expressão citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina. Também foi verificada fraca correlação negativa e estatisticamente significativa (r = -0,499; p = 0,049) entre a expressão núcleo-citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina nos AM. Além disso, uma moderada correlação positiva e estatisticamente significativa entre a expressão estromal da CLIC4 e a expressão da α-SMA nos AM (r = 0,648; p = 0,007) e nos CO (r = 0,541; p = 0,014). Foi observada forte correlação negativa e estatisticamente significativa (r = -0,813; p < 0,001) entre a expressão da E-caderina e a expressão da vimentina nos AM. Os resultados deste estudo sugerem um potencial envolvimento de CLIC4 no processo de transdiferenciação de miofibroblastos, e que a presença destas células é mais frequentemente associada a lesões de comportamento biológico mais agressivo como os AM e CO, além de uma possível atuação desta proteína na regulação do ciclo celular e na TEM nas lesões estudadas (AU).

Benign epithelial odontogenic lesions constitute a heterogeneous group of lesions. the CLIC4 protein acts in the regulation of growth arrest and apoptosis processes, also participating in the process of transdifferentiation of fibroblasts Into myofibroblasts that begin to express α-SMA. Furthermore, CLIC4 expression can interfere with the epithelialmesenchymal transition (EMT) process in neoplasms. This work evaluated the immunoexpression of CLIC4, α-SMA, e-cadherin and vimentin in ameloblastomas (AM) (n = 16), odontogenic keratocysts (OK) (n = 20) and adenomatoid odontogenic tumors (AOT) (n = 8). The analysis of the immunohistochemical expression of the proteins CLIC4, ecadherin and vimentin in the epithelial component of the lesions and of CLIC4 and α-SMA in the connective tissue was carried out in a semi-quantitative way by a previously calibrated evaluator. Expression in the epithelial component of CLIC4 was analyzed separately in the nucleus and cytoplasm, as well as e-cadherin labeling, which was evaluated in the membrane and cytoplasm. Comparisons of the percentages of immunoreactivity in relation to the studied groups were carried out using the nonparametric kruskal-wallis and mann-whitney tests. Possible correlations between the expression of CLIC4, α-SMA, e-cadherin and vimentin were evaluated using the spearman correlation test. The significance level was set at 5% (p < 0.05). Different staining patterns were observed between the groups analyzed, observing that the exclusively cytoplasmic immunoexpression of CLIC4 in the epithelial component of AM (p < 0.001) and AOT (p < 0.001) was significantly higher than that of OK, not demonstrating statistical significance between the AM and AOT. The immunoexpression (nuclear and cytoplasmic) of CLIC4 in the co epithelial lining was significantly higher than that found in the epithelial component of AM (p < 0.001) and AOT (p < 0.001). Stromal CLIC4 immunoexpression was significantly higher in AM (p = 0.009) and OK (p = 0.004) when compared to AOT. The immunoexpression of α-SMA is significantly higher in AM (p = 0.016) and OK (p = 0.034) when compared to AOT. For e-cadherin membrane immunoexpression in co was significantly higher compared to that found in AM (p = 0.009) and AOT (p = 0.024). Greater immunoexpression of e-cadherin (membrane and cytoplasmic) was observed in OK, when compared to AM (p < 0.001) and AOT (p < 0.001). Cytoplasmic ecadherin expression was significantly higher in AM and AOT (p < 0.001) when compared to OK. A statistically significant difference in vimentin immunoexpression was observed between cases of AM and cases of AOT (p = 0.038) and OK (p < 0.001), as well as between AOT and OK (p < 0.001). The correlations tested between the scores of the proteins studied showed that in the am group it was possible to demonstrate a moderate positive and statistically significant correlation (r = 0.527; p = 0.036) between the cytoplasmic expression of clic4 and the cytoplasmic expression of e-cadherin. A weak and statistically significant negative correlation (r = -0.499; p = 0.049) was also found between the nucleus-cytoplasmic expression of clic4 and the cytoplasmic expression of e- cadherin in AM. Furthermore, a moderate positive and statistically significant correlation between the stromal expression of CLIC4 and the expression of α-SMA in AM (r = 0.648; p = 0.007) and OK (r = 0.541; p = 0.014). Additionally, a strong negative and statistically significant correlation (r = -0.813; p < 0.001) was observed between the expression of ecadherin and the expression of vimentin in AM. The results of this study suggest a potential involvement of CLIC4 in the myofibroblast transdifferentiation process, and that the presence of these cells is more frequently associated with lesions with more aggressive biological behavior such as AM and OK, in addition to a possible role of this protein in the regulation of cell cycle and EMT in the lesions studied (AU).

Effects of Xenon on expression of CLIC4 mRNA in neonatal rats brain with white matter damage / 中华实用儿科临床杂志

Objective To investigate the mechanism of white matter damage (WMD) and the neuroprotective effect of Xenon on neonates with WMD.Methods Three-day-old SD rat pups (n =96) were randomly divided into the blank control group (n =24),the WMD control group (n =24),the Xenon intervention group A (n =24) and the Xenon intervention group B (n =24) by random number method according to their birth time.WMD rat models were successfully established by giving intraperitoneal injection of lipopolysaccharide(LPS) 0.05 mg/kg combined with carotid artery ligation and hypoxia for 1 hour in the WMD control group and the Xenon intervention groups.In the control group,only 9 g/L saline (0.05 mg/kg) was injected intraperitoneally,while carotid artery ligation and hypoxia were not administered.Rats in Xenon intervention group A and group B were given inhalation of 500 mL/L Xenon for 3 hours at 0 and 2 hours respectively after establishment of the models.Six rats in each group were randomly selected and decapitated at 0,24,48 and 72 hours after the intervention.The brain white matter on the right was analyzed by using HE staining and myelin basic protein(MBP) immunofluorescence staining,and real-time quantitative polymerase chain reaction was used to detect the expressions level of CLIC4 mRNA.Results (1) Brain tissue pathology:compared with the blank control group,the brain white matter on the right of the WMD control group and the Xenon intervention group A and group B had loose and disordered structure,nuclear pyknosis and cytoplasm loosening.However,the lesions in both Xenon intervention group A and group B were significantly less than those in the WMD control group,and there was no significant difference between the Xenon intervention group A and group B.(2) MBP measurement:the number of MBP-positive cells in the brain white matter on the right of WMD control group was significantly lower than that in the blank control group,while compared with WMD control group,they were significantly higher in Xenon intervention group A and group B.(3) CLIC4 mRNA expression level:compared with blank control group,the expressions levels of CLIC4 mRNA at most time point were higher both in the WMD control group and the Xenon intervention group A and group B (all P ＜ 0.05),except the time point 24 h in the Xenon intervention group A.The expressions of CLIC4 mRNA in group A and group B were significantly decreased compared with those in the WMD control group (all P ＜ 0.05).However,there were no significant differences between Xenon intervention group A and group B (P ＞ 0.05).Conclusions The expressions of CLIC4 mRNA in brain tissues on neonatal rats with WMD significantly increased,indicating that the mitochondrial pathway could be one of the pathological processes of WMD.Early Xenon intervention may reduce neonatal WMD by reducing the expression of CLIC4 mRNA,which plays a neuroprotective role.

Estudo da expressão de CLIC4 e proteínas associadas em queilites actínicas e carcinomas de células escamosas de lábio inferior / Study of the expression of CLIC4 and associated proteins in actinic cheilitis and lower lip squamous cell carcinomas

A proteína do canal de cloro intracelular 4 (CLIC4) regula a passagens dos íons de cloro e relaciona-se com a proteína p53, fator de necrose tumoral α (TNF-α), fator de crescimento transformante-ß (TGF-ß) e com a diferenciação de fibroblastos em miofibroblastos (-SMA) em alguns cânceres humanos. O objetivo desse estudo foi analisar a expressão imuno-histoquímicade CLIC4 e proteínas associadas em queilitesactínicas (QA) e carcinomas de células escamosas de lábio inferior (CCELI), bem como verificar a relação destas entre si e com as características clínicas e morfológicas das lesões.A amostra foi composta de 50casos de QAs e 50de CCELIs com dados clínicos, que inicialmente foram submetidos ao estudo morfológico para sua gradação do risco de transformação maligna (sistema binário) e do grau histológico de malignidade (Bryne, 1992), respectivamente.Todos os casos foram submetidos ao método da imunoperoxidase usando os anticorpos paraCLIC4, p53, TGF-, TNF- e - SMA, os quais foram submetidos à análise semiquantitativa, com exceção de p53, que inicialmente foi analisado de forma quantitativa e em sequência categorizada como as demais. Para a análise da expressão de CLIC4 foi considerada sua localização celular.Comparações das imunomarcações com os parâmetros clínicos e morfológicos das lesões foram realizadas pelo teste U de Mann-Whitney e o coeficiente de Spearmanfoi calculado para avaliar correlações entre as proteínas. A expressão nuclear da CLIC4 e TGF-ß estava aumentadaem QAs de baixo risco, quando comparada ao grupo de alto risco (p<0.0001), enquantoCLIC4 citoplasmática,p53 e TNF-α exibiram maior expressão em QAs de alto risco (p<0.05). No que diz respeito às características clínicas e morfológicas dos CCELIs, a expressão de CLIC4 citoplásmatica foi maior nos casos apresentando metástase linfonodal, casos com estágios clínicos mais avançados ou com alto grau de malignidade (p = 0,005; p = 0,029; p<0,0001). A expressão de p53 foi maior em CCELIs de alto grau de malignidade (p= 0,001) e a TGF-ß diminuiu significativamente conforme o avanço do estágio clínico e do grau histológico dos tumores (p< 0,05).As QAs exibiram uma expressão aumentada de CLIC4 (no núcleo, ou núcleo e citoplasma) e TGF-ß, comparadas aos CCELIs (p < 0,0001). Em contraste, houve aumento na marcação de CLIC4 citoplasmática e α-SMA nos casos de CCELI, quando comparados às QAs (p < 0,0001).Nas QAs observou-se correlação negativa entre a expressão de CLIC4 nuclear ea CLIC4 citoplasmática (r = -0,554; p = <0,0001), e entre a marcação de TGF-ß e α-SMA (r = -0,309; p = 0,029). Nos carcinomas, a expressão de p53 exibiu correlação positiva com TNF-α (r = 0,528; p = 0,0001) e αSMA (r = 0,435; p = 0,002).Os nossos resultados sugeremque uma mudança no padrão de expressão nuclear para citoplasmática de CLIC4 está envolvida no processo decarcinogênese labial, acompanhada de alterações na expressão de p53, TGF-ß, TNF-α e α-SMA, e se relacionamcom alguns dos aspectos morfológicos e clínicos das QAs e CCELIs (AU).

The intracellular chloride channel protein 4 (CLIC4) regulates chloride ions and is related to p53, tumor necrosis factor α (TNF-α), transforming growth factor-ß (TGFß), and with the differentiation of fibroblasts in myofibroblasts (-SMA) in some human cancers. The objective of this study was to analyze the immunohistochemical expression of CLIC4 and associated proteins in actinic cheilites (AC) and squamous cell carcinomas of the lower lip (SCCLL), as well as to verific their relationship with each other and with the clinical and morphological characteristics of the lesions . The sample consisted of 50 cases of AC and 50 of SCCLL with clinical data, which were initially submitted to the morphological study for their gradation of malignant transformation risk (binary system) and histological grade of malignancy (Bryne, 1992). All cases were submitted to the immunoperoxidase method using CLIC4, p53, TGF-ß, TNF- and - SMA antibodies, which were submitted to semiquantitative analysis, except for p53, which was initially analyzed quantitatively and a categorized sequence like the others. For the analysis of CLIC4 expression, its cellular location was considered. Comparisons of the immunoblots with the clinical and morphological parameters of the lesions were performed by the Mann-Whitney U test and the Spearman coefficient was calculated to evaluate correlations between the proteins. Nucleic expression of CLIC4 and TGF-ß was increased in low-risk AC compared to high-risk group (p <0.0001), whereas cytoplasmic CLIC4, p53 and TNF-α showed higher expression in high-risk AC (p < 0.05). As regards the clinical and morphological characteristics of SCCLL, the expression of cytoplasmic CLIC4 was higher in cases presenting lymph node metastasis, cases with more advanced clinical stages or with a high degree of malignancy (p = 0.005, p = 0.029, p <0, 0001). Expression of p53 was higher in highgrade malignant SCCLL (p = 0.001) and TGF-ß decreased significantly as the clinical stage progressed and tumor grade histologically (p <0.05). Increased CLIC4 (in the nucleus, or nucleus and cytoplasm) and TGF-ß, compared to SCCLL (p <0.0001). In contrast, there was an increase in the labeling of cytoplasmic CLIC4 and α-SMA in SCCLL cases, when compared to AC (p <0.0001). In the AC, a negative correlation was observed between nuclear CLIC4 expression and cytoplasmic CLIC4 (r = -0.554, p = <0.0001), and between TGF-ß and α-SMA (r = -0,309; = 0.029). In carcinomas, p53 expression exhibited a positive correlation with TNF-α (r = 0.528, p = 0.0001) and αSMA (r = 0.435, p = 0.002). Our results suggest that a change in CLIC4 cytoplasmic nuclear expression pattern is involved in the process of lip carcinogenesis, accompanied by changes in the expression of p53, TGF-ß, TNF-α and α-SMA, and are related to some of the morphological aspects and clinicians of AC and SCCLL (AU).