Mechanism of Yes-Associated Protein 1 Ameliorating Aristolochic Acid 1-Induced Liver Injury in Mice Based on Untargeted Metabolomics Techniques / 中药新药与临床药理

Objective To investigate the mechanism of Yes-associated protein 1(YAP1)ameliorating aristolochic acid 1(AAI)-induced liver injury in mice based on untargeted metabolomics techniques.Methods There were 83-week-old male hepatocyte-specific Yap1 gene knockout mice(genotyped as Yap1Flox/Flox,Albumin-Cre,aka.Yap1LKO)were randomly selected as the Yap1LKO+AAI group,and 8 Yap1Flox control mice as the Yap1Flox+AAI group.Both groups were injected intraperitoneally with AAI at a dose of 2.5 mg·kg-1·d-1 for 14 consecutive days.Genotypes were identified by tail PCR;serum alanine transaminase(ALT)and aspartate transaminase(AST)activities were determined by microplate assay;histopathological changes of liver tissue were observed by HE staining;and the protein expression of YAP1 in liver tissue was determined by immunohistochemistry.The untargeted metabolomics approach was used to analyze the liver tissue differential metabolites,and the samples were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry,and the differential metabolites were screened by principal component analysis(PCA),Partial least square-discriminant analysis(PLS-DA),and orthogonal partial least squares-discriminant analysis(OPLS-DA);using HMDB database and METLIN database to identify metabolites,and the pathway enrichment of differential metabolites was analyzed by KEGG database.Results(1)After 14 days of AAI induction,the increase of body mass in Yap1LKO mice was lower than that in Yap1Flox mice,but there was no statistical significance(P＞0.05).On day 14,compared with the Yap1Flox+AAI group,the serum ALT and AST enzyme activities in the Yap1LKO+AAI group of mice were significantly increased(P＜0.05),and the histopathological damage of the liver was significantly aggravated.The livers of the Yap1Flox mice had a positive protein expression of YAP1,whereas the Yap1LKO mice did not have a positive protein expression of YAP1.(2)A total of 139 differential metabolites with significant changes(VIP＞1 and P＜0.05)were screened by metabonomic analysis;compared with Yap1LKO+ AAI group,62 liver metabolites in Yap1Flox+AAI group were up-regulated,including choline,taurine,hypotaurine,α-linolenic acid,eleostearic acid,chenodeoxycholic acid and so on.Seventy-seven metabolites were down-regulated including glycerophosphocholine,L-phosphatidylcholine,L-glutamine,L-serine,L-glutathione,5-methionine,phenylalanine,glucose 6-phosphate,lactic acid,uric acid glycosides,etc..KEGG-enriched pathways were mainly choline metabolism,glycerophospholipid metabolism,insulin resistance,glutathione metabolism,etc..Conclusion Hepatocyte-specific Yap1 gene knockout exacerbated AAI-induced liver injury in mice,and YAP1 was involved in the regulation of choline metabolism and glycerophospholipid metabolism through the up-regulation of unsaturated fatty acids,such as choline and taurine,which ameliorated AAI-induced liver injury in mice.

Análise imuno-histoquímica da Yes-Associated Protein (YAP) e sua influência sobre a proliferação celular e a apoptose em lesões odontogênicas epiteliais benignas / Immunohistochemical analysis of Yes-Associated Protein (YAP) and its influence on cell proliferation and apoptosis in benign epithelial odontogenic lesions

Introdução: Os cistos e tumores odontogênicos são lesões que apresentam comportamento biológico heterogêneo e patogênese ainda não totalmente esclarecida. A Yes-associated protein (YAP) atua como um regulador transcricional de genes envolvidos na proliferação celular e na apoptose, participando da ativação de vias associadas ao crescimento cístico e à progressão neoplásica. Objetivo: Analisar a expressão imuno-histoquímica da proteína YAP e correlacioná-la com marcadores envolvidos na proliferação celular e na apoptose em lesões odontogênicas epiteliais benignas. Metodologia: A amostra consistiu de 95 casos de lesões odontogênicas - 25 cistos dentígeros (CDs), 30 CO não sindrômicos (COs), 30 AMB convencionais (AMB-Cs) e 10 AMB unicísticos (AMB-Us) -, além de 10 espécimes de folículo dentários (FD). Foi realizada coleta dos dados clinico-demográficos dos casos, bem como análise morfológica para melhor caracterização da amostra. Os cortes histológicos foram submetidos à técnica imuno-histoquímica através da utilização dos anticorpos YAP, ciclina D1, Ki-67 e Bcl-2, e a análise da expressão destes foi realizada quali-quantitativamente, mediante metodologia adaptada. Os dados coletados seguiram para análise descritiva e estatística (p ≤ 0,05). Resultados: Houve discreta predileção por mulheres (n = 55; 57,6%) e por indivíduos na faixa etária dos 21 aos 40 anos (n = 50; 47,6%), sendo a região posterior de mandíbula mais afetada (64%). A análise da imunoexpressão de YAP revelou maiores níveis de expressão em COs, especialmente nas camadas basal e parabasal, seguido dos AMB-Us e AMB-Cs, que demonstraram moderada imunorreatividade, predominantemente nas células periféricas. Além disso, houve diferenças significativas quanto à imunoexpressão de YAP entre os grupos analisados, com existência de correlações positivas e estatisticamente significativas entre YAP e ciclina D1 em CDs e AMB-Us, e entre YAP e Ki-67 em AMB-Us (p < 0,05). Todavia, entre a imunoexpressão YAP e Bcl-2, foi verificada ausência de correlação estatisticamente significativa. Conclusões: A YAP pode exercer influência sobre a proliferação celular do epitélio de cistos e tumores odontogênicos, auxiliando, assim, na progressão das diferentes lesões odontogênicas (AU).

Background: Odontogenic cysts and tumors present heterogeneous biological behavior, and their etiopathogenesis is not fully understood yet. Yes-associated protein (YAP) acts as a transcriptional regulator of genes involved in cell proliferation and apoptosis, activating pathways associated with cystic growth and neoplastic progression. Objective: To analyze the immunohistochemical expression of YAP protein and correlate it with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions. Methods: The sample consisted of 95 cases of odontogenic lesions - 25 dentigerous cysts (DCs), 30 non-syndromic odontogenic keratocyst (OKCs), 30 conventional AMB (C-AMBs), and 10 unicystic AMB (UAMBs) -, in addition to 10 specimens of dental follicles (DF). Clinicodemographic data collection was carried out, as well as morphological analysis for better characterization of the sample. The histological sections were submitted to the immunohistochemical technique using YAP, cyclin D1, Ki-67, and Bcl-2 antibodies, and their immunoexpression analysis was performed qualitatively and quantitatively, through an adapted methodology. The collected data were submitted for descriptive and statistical analysis (p ≤ 0.05). Results: There was a slight predilection for women (n = 55; 57.6%) and individuals aged between 21 and 40 years (n = 50; 47.6%), with the posterior region of the mandible as the most affected site (64%). Analysis of YAP immunoexpression revealed higher expression levels in OKCs, especially in the basal and parabasal layers, followed by U-AMBs and C-AMBs, which showed moderate immunoreactivity, predominantly in peripheral cells. In addition, there were significant differences in YAP immunoexpression between the analyzed groups, with positive and statistically significant correlations between YAP and cyclin D1 in DCs and U-AMBs, and between YAP and Ki-67 in U-AMBs (p < 0.05). However, between YAP and Bcl-2 immunoexpression, there was no statistically significant correlation. Conclusions: YAP may influence on the cell proliferation of odontogenic cysts and tumors epithelium, thus helping with the progression of the different odontogenic lesions (AU) .

Role of YAP/OPA1 signaling pathway in propofol-induced reduction of oxygen-glucose deprivation and restoration injury in hippocampal neurons / 中华麻醉学杂志

Objective:To evaluate the role of Yes-associated protein (YAP)/Optic atrophy-1 (OPA1) signaling pathway in propofol-induced reduction of oxygen-glucose deprivation and restoration(OGD/R) injury in hippocampal neurons.Methods:HT22 mouse hippocampal neurons at the logarithmic growth phase were divided into 4 groups ( n=54 each) using a random number table method: control group (group C), group OGD/R, propofol group (group P) and propofol + YAP silencing group (group P + siRNA-YAP). The cells were subjected to O 2-glucose deprivation for 6 h followed by restoration of O 2-glucose supply for 24 h. In group P, propofol 50 μmol/L was added immediately after restoration of O 2-glucose supply. In P+ siRNA-YAP group, siRNA-YAP was transfected at 48 h before model preparation. The viability of neurons was measured by CCK-8 assay, ROS content and apoptosis rate were measured by flow cytometry, the content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) were determined by spectrophotometry, the content of mitochondrial ATP was determined by fluorescein fluorescence method, the nuclear translocation of YAP was observed by immunofluorescence, and the expression of YAP, phosphorylated YAP (p-YAP) and OPA1 was detected by Western blot. Results:Compared with group C, the viability of hippocampal neurons was significantly decreased, the contents of ROS and MDA and apoptosis rate were increased, the SOD activity, MMP and mitochondrial ATP content were decreased, the expression of p-YAP protein was up-regulated, OPA1 expression was down-regulated ( P<0.05), and the fluorescence intensity of YAP in nucleus was weakened in group OGD/R. Compared with OGD/R group, the viability of neurons was significantly increased, the contents of ROS and MDA and apoptosis rate were decreased, the activity of SOD, MMP and content of mitochondrial ATP were increased, the expression of p-YAP protein was down-regulated, the expression of OPA1 protein was up-regulated( P<0.05), and the fluorescence intensity of YAP in nucleus was enhanced in P group. Compared with group P, the viability of neurons was significantly decreased, the contents of ROS and MDA and apoptosis rate were increased, the SOD activity, MMP and mitochondrial ATP content were decreased, the expression of p-YAP, YAP and OPA1 was down-regulated ( P<0.05), and the fluorescence intensity of YAP in nucleus was weakened in group P+ siRNA-YAP. Conclusions:The mechanism by which propofol reduces OGD/R injury in hippocampal neurons may be related to activation of YAP/OPA1 signaling pathway.

Regulation of Yes associated protein in diabetic nephropathy mice by Yishen Huashi Granule / 中国药理学通报

Aim To explore the protective effect of Yishen Huashi Granule (YSHS) on streptozotocin (STZ) - indueed diabetes nephropathy (DN) in mice and its possible mechanism. Methods The STZ induced DN mice model was established, which was randomly divided into model group, YSHS group and YAP inhibitor Vertepofin group, and the eontrol group was also established. The intervention was started eight weeks after the successful modeling with the course of four weeks. Urine protein concentration before and after intervention in each group as well as serum creatinine (Scr) and blood urea nitrogen (Bun) levels after intervention were measured. After the treatment, the mice were sacrificed, and the pathological changes of glomeruli were observed by light microscope HE staining. The protein expression of YAP, p-YAP S127 and CTGF were detected by Western blot, and the mRNA expressions of YAP, CTGF and podocyte functional proteins nephrin, podophyxin and WT1 were detected by RT-PCR. Results The biochemical indexes of YSHS group were better than those of model group, and HE staining showed that the pathological injury of glomeruli was improved. In the model group the protein expression of YAP, p-YAP (S127) and CTGF as well as the mRNA expression of YAP and CTGF increased, while the mRNA expression of nephrin, podocalyxin and WT1 decreased. After YSHS treatment, the protein expression of YAP, p-YAP S127, CTGF and the mRNA expression of YAP and CTGF decreased, while the mRNA expression of nephrin, podocalyxin and WT1 increased. Conclusions YSHS can reduce urinary protein, protect renal function and alleviate glomerular pathological injury in DN mice. Its possible mechanism may be related to the down-regulation of YAP in renal tissue, the reduction of CTGF expression level and the up-regulation of podocyte protein mRNA expression.

Activity and mechanism of palbinone against hepatic fibrosis and inflammation / 药学学报

The study aims to investigate the anti-hepatic fibrosis and anti-inflammatory activities of palbinone, and to explore the internal regulatory mechanism, so as to lay an active foundation for its development as an anti-non-alcoholic steatohepatitis (NASH) candidate. First, sulforhodamine B (SRB) method was used to detect the effect of palbinone on the proliferation of human hepatic stellate cells LX-2 and rat hepatic stellate cells HSC-T6. Following, in the in vitro hepatic fibrosis cell model that activated by transforming growth factor beta 1 (TGF-β1), quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the inhibitory effect of different concentrations of palbinone on the transcription level and protein expression level of hepatic fibrosis markers. And the regulating mechanism of palbinone on fibrosis-related genes was analyzed at the same time. In addition, in the inflammatory cell model that induced by lipopolysaccharide (LPS) and nigericin, ELISA was used to detect the effect of palbinone on the released interleukin-1β (IL-1β) level. At the same time, Western blot was used to detect the effect of palbinone on the related proteins of inflammatory pathway. The results showed that palbinone could significantly inhibit the proliferation activity of LX-2 and HSC-T6, and their half maximal inhibitory concentration (IC50) values ​​were (375.11 ± 55.45) and (260.27 ± 36.81) nmol·L-1, respectively. In addition, palbinone showed a dose-dependent inhibitory effect on the expression levels of TGF-β1-induced fibrosis-related genes, including collagen type Ⅰ α 1 (COL1A1), TGF-β1, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase 1 (TIMP1). Mechanism study showed that palbinone may decrease the expression level of Yes-associated protein (YAP), thereby weakening its activation effect on the downstream fibrosis pathway. In addition, palbinone also exerted an anti-inflammatory effect by inhibiting the activity of nuclear factor kappa-B (NF-κB) signaling pathway and reducing inflammatory factors cysteinyl aspartate specific proteinase-1 (caspase-1) and IL-1β release. In conclusion, palbinone can not only inhibit the proliferation and activation of hepatic stellate cells by inhibiting the expression of YAP, but also inhibit the expression and release of inflammatory factors at the same time. All these studies provide theoretical support for the development of palbinone as an anti-nonalcoholic steatohepatitis drug.

Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP / 中国中药杂志

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

YAP regulates the liver size during the fasting-refeeding transition in mice

Liver is the central hub regulating energy metabolism during feeding-fasting transition. Evidence suggests that fasting and refeeding induce dynamic changes in liver size, but the underlying mechanisms remain unclear. Yes-associated protein (YAP) is a key regulator of organ size. This study aims to explore the role of YAP in fasting- and refeeding-induced changes in liver size. Here, fasting significantly reduced liver size, which was recovered to the normal level after refeeding. Moreover, hepatocyte size was decreased and hepatocyte proliferation was inhibited after fasting. Conversely, refeeding promoted hepatocyte enlargement and proliferation compared to fasted state. Mechanistically, fasting or refeeding regulated the expression of YAP and its downstream targets, as well as the proliferation-related protein cyclin D1 (CCND1). Furthermore, fasting significantly reduced the liver size in AAV-control mice, which was mitigated in AAV Yap (5SA) mice. Yap overexpression also prevented the effect of fasting on hepatocyte size and proliferation. Besides, the recovery of liver size after refeeding was delayed in AAV Yap shRNA mice. Yap knockdown attenuated refeeding-induced hepatocyte enlargement and proliferation. In summary, this study demonstrated that YAP plays an important role in dynamic changes of liver size during fasting-refeeding transition, which provides new evidence for YAP in regulating liver size under energy stress.

Mechanism of regulatory effect of recombinant human HMGB1 on endothelial cell angiogenesis / 器官移植

Objective To unravel the possible mechanism of the role of recombinant human high mobility group box 1 (rhHMGB1) protein in regulating the angiogenesis of endothelial cells. Methods Endothelial cells were divided into the control group, bone marrow mesenchymal stem cells (MSC) supernatant group and rhHMGB1 group. The proliferation and survival of endothelial cells were detected by cell counting kit(CCK)-8 assay. The relative expression levels of vascular endothelial growth factor (VEGF), Yes-associated protein (YAP), CD31 and hypoxia inducible factor (HIF)-1α proteins were determined by Western blot. The relative expression levels of VEGF, YAP, CD31 and HIF-1α messenger RNA (mRNA) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The migration ability of endothelial cells was assessed by Transwell chamber test. The localization of YAP was detected by immunofluorescence staining. Results Compared with the control group, the migration rate of endothelial cells was increased in the rhHMGB1 group (P < 0.05), and the cell migration rate was enhanced over time. Compared with the control group, the relative expression levels of VEGF and p-YAP proteins were up-regulated in the MSC supernatant group, and the differences were statistically significant (both P < 0.05). Compared with the control group, the relative expression levels of VEGF and HIF-1α proteins, VEGF and CD31 mRNA and YAP and p-YAP proteins were up-regulated, and YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Compared with the MSC supernatant group, the relative expression levels of CD31 mRNA and YAP protein were up-regulated, and the YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Conclusions Exogenous high-concentration rhHMGB1 may promote the migration ability of endothelial cells and up-regulate the expression levels of angiogenesis-related proteins by regulating the recruitment of YAP to the nucleus.

β-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis

Abstract Objective: To explore whether the effect of β-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. Methods: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. Results: β-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate β-catenin expression in MI cardiac tissue. β-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas β-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of β-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in β-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of β-catenin overexpression on cell survival and apoptosis. Conclusions: The present data indicate that β-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.

Mechanism and clinical significance of YAP in the progression of gastric cancer / 国际肿瘤学杂志

Gastric cancer is one of the common malignant tumors with a high incidence in China. With the deepening of the research on tumor molecular biology, molecular targeted therapy has shown its advantages, and immunotherapy has attracted much attention. Yes-associated protein (YAP) is an effector protein that plays a major role in Hippo pathway, and it can regulate the growth of cells after binding with transcription factors in the nucleus. In recent years, many studies have shown that the high expression of YAP is closely related to the occurrence, development and metastasis of gastric cancer, and may be an important target of chemotherapy resistance. The further study can provide reference for clinical treatment.

Yes-associated protein ameliorates tumor necrosis factor-α-induced epithelial barrier dysfunction by affecting the F-actin / 中国小儿急救医学

Objective:To investigate the protective role of Yes-associated protein(YAP)in intestinal epithelial barrier injury.Methods:The intestinal epithelial barrier model was established by culturing human colorectal adenocarcinoma cell line Caco-2 cells, which were divided into four groups: control group, Caco-2 monolayers did not receive any treatment; recombinant human tumor necrosis factor-α(rhTNF-α)group, 100 μg/L of rhTNF-α was added to Caco-2 monolayers; vector+ rhTNF-α group, Caco-2 monolayers were first added with control plasmid pcDNA3.1-vector, and 100 μg/L rhTNF-α was added 24 hours later; YAP+ rhTNF-α group, Caco-2 cells with barrier construction were first added with pcDNA3.1-YAP, and 100 μg/L rhTNF-α was added 24 hours later.Realtime-PCR and Western blot were used to evaluate YAP mRNA and protein expression level.Epithelial permeability was assayed by trans-epithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran 40(FD-40 flu). Cellular distribution of F-actin was assayed by immunofluorescence staining.Results:Compared with control group[(607.3±29.3)Ω·cm 2], TEER of rhTNF-α group[(265.3±32.7)Ω·cm 2] decreased, while TEER of YAP+ rhTNF-α group[(387.0±18.7)Ω·cm 2]increased compared with rhTNF-α group, the differences were statistically significant( P<0.001). The FD-40 flux of rhTNF-α(22.7%±0.5%) group was higher than that of the control group(6.3%±0.9%), while the FD-40 flux of Yap + rhTNF-α group(12.2%±0.8%) was lower than that of rhTNF-α group, the differences were statistically significant( P<0.001). Immunofluorescence staining showed that compared with the control group, the cytoskeletal F-actin fiber dense spot decreased in rhTNF-α group, and some cells showed obvious trans-cellular stress fiber structure, while the peripheral actin band was clear in YAP+ rhTNF-α group, and the intracellular stress fiber decreased.YAP+ TNF-α group appeared as a clear, peripheral actin ribbon with a decrease in cytoplasmic stress fibres. Conclusion:YAP overexpression significantly inhibits TNF-α induced decline of TEER, and increases of FD-40 flux and F-actin rearrangement of Caco-2.YAP could ameliorate TNF-α induced intestinal epithelial barrier injury by regulating cytoskeleton F-actin.

Mechanism study of neurofibromin 1 in gallbladder cancer / 中华消化外科杂志

Objective:To investigate the mechanism of neurofibromin 1 (NF1) in gallbla-dder cancer.Methods:The experimental study was conducted. Human gallbladder cancer cell lines, including GBC-SD, NOZ, SGC996, EH-GB1, ZJU0428, human embryonic kidneys cell line 293T and human cervical cancer cell line HELA, were cultured. The recombinant plasmids (mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA) were constructed for co-immunoprecipitation experiment. The truncated Yes associated protein 1(YAP1) and NF1 recombinant proteins were purified in vitro. The interaction between NF1 and YAP1 in vitro or in vivo were verified by isothermal titration calori-metry (ITC) assay, GST pull-down experiment, co-immunoprecipitation, immunofluorescence, laser confocal microscopy, and the expression of NF1 protein in different gallbladder cancer cell lines was verified by Western blot experiments. Observation indicators: (1) interaction between NF1 and YAP1 in vitro; (2) interaction between NF1 and YAP1 in cells; (3) expression of NF1 protein in different human gallbladder cancer cell lines. The dissociation constants were exported from ITC 200 software and represented as Mean± SD. Count data were represented as absolute numbers. Results:(1) Interaction between NF1 and YAP1 in vitro. ① Results of ITC assay showed that there was interac-tion between PPQY and YAP1-WW1, between PPQY and YAP1 (Amino acid residues 162?275), and the dissociation constants between PPQY and YAP1-WW1, between PPQY and YAP1(Amino acid residues 162?275) were (0.42±0.06)mmol/L, (0.69±0.14)mmol/L, respectively. ② GST pull-down results indicated that the target protein His-Sumo-YAP1 WW1 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW1, relative to the reaction system between GST protein and His-Sumo-YAP1 WW1. The target protein His-Sumo-YAP1 WW2 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW2, relative to the reaction system between GST protein and His-Sumo-YAP1 WW2. (2) Interaction between NF1 and YAP1 in cells. ① Co-immunoprecipitation results indica-ted that NF1 protein was observed in cell lysis solution which was incubated by FLAG gel beads and cotransfected with mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA. ② Immuno-fluorescence and laser confocal microscopy results indicated that YAP1 and NF1 with obvious fluorescence were co-localized in the cytoplasm of human gallbladder cancer NOZ cells. However, YAP1 with obvious fluorescence was localized in the nucleus of human gallbladder SGC996 cells and NF1 showed weak fluorescence. (3) Expression of NF1 protein in different human gallbladder cancer cell lines. Western blot results showed that with the expression level of NF1 protein in HELA cell line as the standard, the relative expression levels of NF1 protein in EH-GB1, GBC-SD, NOZ, SGC996, ZJU0428 cell lines were 1.28, 0, 1.01, 0, 0, respectively. Conclusion:NF1 affects the gallbladder cancer by directly acting on YAP1 protein.

Role of Yes-associated protein 1 in acute lung injury in septic mice: the relationship with ferroptosis / 中华麻醉学杂志

Objective:To evaluate the role of Yes-associated protein 1 (YAP1) in acute lung injury (ALI) and the relationship with ferroptosis in septic mice.Methods:Twenty-four male wild-type mice and 24 YAP1 conditional knockout mice, aged 9-10 weeks, weighing 22-25 g, were divided into 2 groups ( n=12 each) using a random number table method: wild-type sham operation group (WT+ Sham group) and wild-type sepsis-induced ALI group (WT+ ALI group); YAP1 conditional knockout sham operation group (CKO+ Sham group) and YAP1 conditional knockout sepsis-induced ALI group (CKO+ ALI group). The sepsis-induced ALI model was developed by cecal ligation and perforation (CLP) in anesthetized animals.The bronchoalveolar lavage fluid (BALF) was collected at 24 h after CLP to determine the protein concentration (by bicinchoninic acid method) and concentrations of interleukin-1beta (IL-1β) and tumor necrosis factor-α (TNF-α) (by enzyme-linked immunosorbent assay). Mice were then sacrificed, and the lung tissues were obtained for examination of ultrastructure (using a transmission electron microscope) and for determination of wet/dry lung weight ratio (W/D ratio), contents of Fe 2+ , malondialdehyde (MDA) and glutathione (GSH) (by colorimetric assay), and expression of YAP1, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) (by Western blot). Results:Compared with WT+ Sham group, the concentrations of protein in BALF, IL-1β and TNF-α were significantly increased, W/D ratio and contents of Fe 2+ and MDA were increased, GSH contents were decreased, the expression of GPX4 and SLC7A11 was down-regulated, ACSL4 expression was up-regulated ( P<0.05), alveolar epithelial cells showed characteristic changes of ferroptosis with mitochondrial shrinkage and decreased mitochondrial cristae in WT+ ALI group.Compared with WT+ CLP and CKO+ Sham groups, the concentrations of protein in BALF, IL-1β and TNF-α were significantly increased, W/D ratio and contents of Fe 2+ and MDA were increased, GSH contents were decreased, the expression of GPX4 and SLC7A11 was down-regulated, ACSL4 expression was up-regulated ( P<0.05), and the mitochondria in alveolar epithelial cells in lung tissues shrank obviously, and the mitochondrial cristae were reduced or even disappeared in CKO+ CLP group ( P<0.05). Conclusions:YAP1 is involved in the endogenous protective mechanism against ALI, which is related to inhibition of ferroptosis in septic mice.

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer / 中南大学学报(医学版)

OBJECTIVES@#Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer.@*METHODS@#We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3.@*RESULTS@#After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC.@*CONCLUSIONS@#YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.

Mild hypothermia alleviates renal ischemia-reperfusion injury by up-regulating the expression of cold-shock protein RBM3 / 器官移植

Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2.

Research progress on the role of Hippo signaling pathway in posterior capsular opacification / 国际眼科杂志(Guoji Yanke Zazhi)

@#Posterior capsular opacification is the most common complication after cataract extraction, which seriously influences the quality of life of patients. At present, there is no effective measure to prevent posterior capsular opacification. Surgery or Nd:YAG laser is often used in clinical, and a new treatment is urgently needed. Hippo signaling pathway is involved in the steady-state regulation of many mammalian cells and organs. Recent studies have shown that Hippo signaling pathway can regulate the proliferation, apoptosis, differentiation and other behaviors of lens epithelial cells. Hippo signaling pathway may provide a new target in the treatment of posterior capsular opacification. This article reviews the composition, regulatory mechanism of Hippo signaling pathway and its application in posterior capsular opacification. In order to provide a broader idea for the prevention and treatment of posterior capsular opacification.

Expression of PKCι/YAP1 in Cervical Cancer and Its Clinical Significance / 肿瘤防治研究

Objective To investigate the role of PKCι, YAP1 and high-risk HPV infection in the local immune microenvironment of cervical cancer. Methods We chose 80 cases of normal tissue of the cervix (NCT), cervical low-grade squamous intraepithelial lesion (LSIL), cervical high-grade squamous intraepithelial lesion (HSIL) and early cervical squamous cell carcinoma (SCC) each. Four groups were collected.The infection rate of high-risk HPV in four groups was determined by real-time fluorescence PCR method. The expression levels of PKCι, YAP1, CD4 and CD8 in four groups were measured and correlated by IHC and clinicopathologic features were also analyzed. Results The differences of high-risk HPV infection rate and PKCι, YAP1, CD4, CD8 positive rate among groups of NCT, LSIL, HSIL and SCC had statistical significance (P < 0.05). The level of cervical lesions was positively associated with high-risk HPV infection and positive PKCι, YAP1, CD8 expression (P < 0.05), while negatively associated with positive CD4 expression (P < 0.05). HPV infection and positive PKCι, YAP1, CD8 expression were positively correlated with each other in SCC, while were all negatively correlated with positive CD4 expression(P < 0.05). The differences of HPV infection, PKCι, YAP1 and CD8 positive expression were significant in different levels of differentiation and vascular invasion of SCC (P < 0.05). Conclusion The patients with cervical lesions are often accompanied by high-risk HPV infection and abnormal expression of PKCι, YAP1, CD4 and CD8, which may have synergistic effects on each other, causing the local immunosuppression microenvironment of SCC. It provides a possible strategy for the study of pathogenesis, diagnosis and treatment of cervical cancer.

Mechanosensitive ion channel PIEZO1 promotes glioma invasion and aggression by regulating Yes-associated protein / 中华神经医学杂志

Objective:To investigate the molecular mechanism of PIEZO1 in promoting the invasion and aggression of glioma via Yes-associated protein (YAP) delivering mechanical signals.Methods:(1) Specimen detection: specimens from 94 patients accepted glioma resection in our hospital from February 2015 to March 2017 were chosen; immunohistochemical staining was used to detect the PIEZO1 expression in these glioma tissues of different grades. (2) Cell experiment: human glioma cell lines U87 and U251 were cultured under different matrix; the expressions of PIEZO1 and YAP were detected by immunofluorescent staining, and the PIEZO1 protein expression was detected by Western blotting. (3) Cell experiment after lentivirus transfection: the U87 and U251 cells were divided into negative control group and sh-PIEZO1 group according to the presence or lack of shRNA lentivirus vector; the role of PIEZO1 in glioma proliferation and invasion was detected by clone formation assay, proliferation and invasion assay, and Western blotting; after PIEZO1 silencing, Western blotting was used to detect the expressions of mechanical signal pathway proteins, YAP, FAK, and β1-integrin; real time-PCR was used to detect the mRNA expressions of CTGF and CYR61, which were downstream target genes of YAP; immunofluorescent staining was used to detect the YAP expression after PIEZO1 silencing. Results:(1) The PIEZO1 expression in glioma specimens increased with glioma grading, and the PIEZO1 expression in IDH wild-type patients was higher than that in IDH mutant patients. (2) The PIEZO1 and YAP expressions increased with the increase of matrix stiffness; as compared with that in the 0.2 kPa group, the PIEZO1 protein expression in stromal cultured cells of 16 and 64 kPa groups was significantly increased ( P<0.05). (3) After PIEZO1 silencing, U87 cells became large and tentacles increased, while U251 cells were mostly with long fusiform; as compared with those in the negative control group, the number of cell clones, and the proliferation rate and invasive cell count at each time point in the sh-PIEZO1 group were significantly smaller/decreased ( P<0.05); as compared with those in the negative control group, the epithelial marker E-cadherin expression was significantly increased, while the expressions of stromal markers Vimentin, Snailin and Slug were significantly decreased in the sh-PIEZO1 group ( P<0.05). Finally, Western blotting and immunofluorescent staining showed that PIEZO1 silence significantly increased the phosphorylated-YAP expression, significantly decreased the YAP nuclear expression, significantly down-regulated the CYR61 and CTGF expressions, and significantly reduced the levels of integrin β1 and phosphorylated-FAK; there were significant differences on these indexes between the negative control group and sh-PIEZO1 group ( P<0.05). Conclusion:PIEZO1 regulates the response of glioma cells to mechanical signal and promotes proliferation and invasion of glioma cells through YAP, which is an attractive therapeutic target for glioma treatment.

Effect of Yes-associated protein in regulation of electroacupuncture pretreatment on cerebral ischemia reperfusion injury rats / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) pretreatment on inflammatory reaction, apoptosis and expression of Yes-associated protein (YAP) of ischemic penumbra of cerebral cortex in cerebral ischemia reperfusion injury rats, and to explore the possible mechanism of its neuroprotection effect.@*METHODS@#A total of 84 SD rats were randomized into a sham operation group (12 rats), a model group (18 rats), an EA group (18 rats), an EA+YAP virus transfection group (18 rats) and an EA+virus control group (18 rats). Except for the sham operation group, thread embolization method was adopted to establish the middle cerebral artery occlusion (MCAO) model in rats of the other groups. EA was applied at "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min in the 3 EA intervention groups 2 h before model establishment, disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in intensity. Adenovirus transfection technique was used to induce gene silencing of YAP in the EA+YAP virus transfection group, and adenovirus vectors was injected as negative control in the EA+virus control group 4 d before model establishment. Twenty-four hours after model establishment, neurological function score was evaluated, the relative cerebral infarction area was observed by TTC staining, the apoptosis in the ischemic penumbra of cerebral cortex was detected by TUNEL staining, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex was detected by ELISA method, the expression of YAP was detected by Western blot and immunofluorescence.@*RESULTS@#Compared with the sham operation group, the expression of YAP was increased in the model group (@*CONCLUSION@#Electroacupuncture pretreatment can effectively improve the ischemia reperfusion injury, its mechanism may be related to up-regulating the expression of YAP in the ischemic penumbra of cerebral cortex and relieving the apoptosis and inflammatory reaction.

Effect and mechanism of YAP in hepatic ischemia-reperfusion injury / 器官移植

Objective To explore the effect and mechanism of Yes-associated protein (YAP) in hepatic ischemia-reperfusion injury (IRI) of mice. Methods Forty male C57BL/6 mice were randomly divided into the sham operation group (Sham group), lysophosphatidic acid (LPA) + Sham group, IRI group and LPA+IRI group, 10 mice in each group. Liver tissue and serum samples were collected at 6 h after ischemia-reperfusion. The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Histopathological changes and macrophage infiltration of liver tissues were detected by hematoxylin-eosin (HE) staining and immunohistochemical staining. The protein expression level of YAP was detected by Western blot. The messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), interleukin (IL)-1 and IL-6 were quantitatively measured by reverse transcription polymerase chain reaction (RT-PCR). Results Western blot results demonstrated that the protein expression level of YAP in the LPA+IRI group was significantly up-regulated than that in the IRI group. Compared with the Sham group, the ALT and AST were significantly higher in the IRI group (both P < 0.05). The serum levels of ALT and AST in the LPA+IRI group were significantly lower than those in the IRI group (both P < 0.05). HE staining revealed that the morphology of hepatocytes was normal in the Sham group and LPA + Sham group. Pathological changes, such as liver congestion, liver cell swelling and structural abnormalities of hepatic lobule, occurred in the LPA+IRI group and IRI group. Compared with the IRI group, pathological changes were alleviated in the LPA+IRI group. RT-PCR indicated that the mRNA expression levels of TNF-α, iNOS, IL-1 and IL-6 in the LPA+IRI group were lower than those in the IRI group (all P < 0.05). Immunohistochemical demonstrated that LPA partially inhibited macrophage infiltration in ischemic tissues after IRI. Conclusions YAP can significantly mitigate hepatic IRI. The mechanism is associated with the regulation of macrophage recruitment and activation.