Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/β-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/β-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/β-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway, inferring that ERK/β-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions. Studies have found that astronauts often have skin problems such as allergic rashes and effusion, a significant reduction in elasticity, obvious thinning of the dermis, increased skin sensitivity, and delayed wound healing ,2,3. OneDermal fibroblasts are critical in supporting skin-repairing . Bone maWnt/β-catenin signaling can participate in the occurrence of fibrosis and regulate the differentiation of BMSCs into lung fibroblasts ,10. HoweTherefore, this study investigated the changes of BMSCs differentiation into dermal fibroblasts under SMG conditions and further revealed the possible molecular mechanism of the changes. Our data suggest that SMG inhibits the dermal fibroblastic differentiation of BMSCs, possibly by suppressing the ERK/β-catenin signaling pathway.BMSCs can be differentiated into fibroblasts with connective tissue growth factor (CTGF) and ascorbic acid treatment ,16,17. FThe 2D-clinostat device A was utiStudies have pointed that Wnt/β-catenin signaling may play a critical role in fibroblastic differentiation . As the Studies have found that the phosphorylation of ERK1/2 is an upstream signal molecule of the Wnt/β-catenin signaling . AdditioIn this study, we have established a differentiation model of BMSCs into dermal fibroblasts and demonstrated that SMG inhibited the dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway. To our knowledge, this is the first study to demonstrate the above findings. Our findings provide a potential target for directing the dermal fibroblastic differentiation of BMSCs to promote skin-repairing for clinical application of skin-repairing in space.Dermal fibroblasts are the main structural components of the skin dermis, which can synthesize and secrete extracellular matrix such as collagen fibers . They arIn recent years, more and more attention has been paid to mechanical stimulation, such as microgravity, in regulating stem cell fate . It has The canonical Wnt/β-catenin signaling pathway regulates cell growth, differentiation, and apoptosis . Sun andTaken together, SMG inhibits the Wnt/β-catenin signaling to impede dermal fibroblastic differentiation of BMSCs, which is possibly due to the decreased phosphorylation of ERK1/2. The upregulation of the Wnt/β-catenin signaling with LiCl treatment reversed the inhibition of SMG on dermal fibroblastic differentiation of BMSCs. Activating the phosphorylation of ERK1/2 by tBHQ improved the Wnt/β-catenin signaling. Our results suggest that both the Wnt/β-catenin signaling and the phosphorylation of ERK1/2 are potential targets for the SMG-inhibited dermal fibroblastic differentiation of BMSCs. An overview schematic diagram  exhibits2 at 37 °C. All of the cells used in this study were between passage 2–4.All animal experimental procedures were performed conforming to ethical standards, and national and international standards, and also were approved by Chongqing Science and Technology Commission, Chongqing, China. All Sprague–Dawley (SD) rats, regardless of their gender, used in this study weighing approximately 100 g were provided by Chongqing Medical University. The BMSCs were isolated from the SD rats by the method of whole bone marrow adhesion, as described previously . BrieflyFor dermal fibroblastic differentiation, when the cells reached 80%-90% confluence, the culture medium was changed to the differentiation medium supplemented with 100 ng/mL recombinant human CTGF  and 50 µg/mL ascorbic acid . The differentiation medium was changed every 2 days for 7 and 14 days.−3 g, and the centrifugal force generated by the 2D-clinostat was approximately 10−3 g too. BMSCs were injected into each chamber at a total count of 3 × 105 cells and all air bubbles were carefully removed to prevent fluid shear stress. After cells were attached to the slide for 24 h and reached 80–90% confluence, the chambers were fixed to the clinostat, and the effect of simulated microgravity was achieved by rotation around the horizontal axis at 10 rpm for 7 days in an incubator with 5% CO2 at 37 °C. As normal gravity control groups, static cells were cultured under the same conditions but were not rotated.In this study, the 2D-clinostat device constructed by the National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, China, was utilized to simulate microgravity. The clinostat is an effective ground tool for simulating microgravity, the principle of clinostat to model simulated microgravity is to continuously rotate and change the orientation of the cell to gravity to prevent the cell from feeling gravity. As shown in Total cellular RNA was extracted using an RNA extraction kit  according to the manufacturer’s instructions. The total extracted RNA concentration was determined using a Nanodrop 2000 spectrophotometer . Next, 1 µg of the extracted RNA was reversely transcribed to cDNA by using the PrimeScript™ RT reagent kit with gDNA eraser . Then, quantitative Real-time PCR was performed using a CFX96™ real-time PCR detection system  with 2x SYBR Green qPCR Master Mix  according to the manufacturer’s instructions. The PCR reaction conditions used were 5 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 30 s at 60 °C. The primer sequences are listed in The total protein of the BMSCs was extracted on ice using a RIPA lysis buffer  supplemented with a 1% protease inhibitor cocktail . Protein concentrations of the lysates were measured with a BCA protein assay kit . The lysates mixed with a 5x sample loading buffer were boiled for 10 min at 100 °C for denaturation. Then, 20 µg of each protein sample was separated with 10% SDS-PAGE gel and was electroblotted onto PVDF membranes . The membranes were blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST). Then the membranes were incubated with primary antibodies , , , , , and  diluted in primary antibody diluents  overnight at 4 °C. Next, the membrane was washed 3 times with TBST for 10 min each time and incubated with an appropriate secondary antibody  with 5% non-fat milk for 1 h at room temperature and then washed 3 times with TBST for 10 min each time, the protein expression was visualized using an ECL blotting analysis system .The BMSCs were washed with PBS, fixed by 4% paraformaldehyde  for 30 min. Then the BMSCs were rinsed with distilled water 3 times, followed by dyeing with Sirius red staining solution  for 1 h. Next, the BMSCs were rinsed with distilled water 3 to 5 times until the color was eluted. Digital images were taken using an inverted phase contrast microscope . After imaging, the expression of Col I and Col III was analyzed and quantified by the software ImageJ.p values were analyzed by one-way analysis of variance (ANOVA) followed by two-tailed Student’s t-test. When p < 0.05, the data between 2 groups was considered to have statistically significant differences.All data were expressed as the mean


Ambystoma mexicanum). Our results showed the higher conservation of axolotl mc3r and mc4r across species than mrap2, especially the transmembrane regions in these proteins. Phylogenetic analysis indicated that the axolotl MC3R/MC4R clustered closer to their counterparts in the clawed frog, whereas MRAP2 fell in between the reptile and amphibian clade. We also identified a clear co-expression of mc3r, mc4r, and mrap2 along with pomc and agrp in the axolotl brain tissue. In the presence of MRAP2, the pharmacological stimulation of MC3R by α-MSH or ACTH significantly decreased. MRAP2 significantly decreased the cell surface expression of MC4R in a dose dependent manner. The co-localization and formation of the functional complex of axolotl MC3R/MC4R and MRAP2 on the plasma membrane were further confirmed in vitro. Dramatic changes of the expression levels of mc3r, mrap2, pomc, and agrp in the fasting axolotl hypothalamus indicated their critical roles in the metabolic regulation of feeding behavior and energy homeostasis in the poikilotherm aquatic amphibian.The Melanocortin-3 receptor (MC3R) and Melanocortin-4 receptor (MC4R), two members of the key hypothalamic neuropeptide signaling, function as complex mediators to control the central appetitive and energy homeostasis. The melanocortin 2 receptor accessory protein 2 (MRAP2) is well-known for its modulation on the trafficking and signaling of MC3R and MC4R in mammals. In this study, we cloned and elucidated the pharmacological profiles of MRAP2 on the regulation of central melanocortin signaling in a relatively primitive poikilotherm amphibian species, the Mexican axolotl ( As a key neuropeptide hormone family, the melanocortins and their receptors have been reported to participate in numerous physiological functions such as energy homeostasis, natriuresis, inflammation, and exocrine secretion , 2. The Five melanocortin receptor subtypes (MC1R–MC5R) have been identified to be distributed in different tissues with segregated biological functions , 8, 9. TIn addition to the four melanocortins  and five MCRs (MC1R–MC5R), the melanocortin system also includes two endogenous antagonists of the MCRs: agouti (ASP)  and agouA pair of GPCR single-transmembrane accessory proteins: melanocortin 2 receptor accessory protein (MRAP) and melanocortin 2 receptor accessory protein 2 (MRAP2) also function in the complex melanocortin regulatory system . MRAP2 cSeveral studies have been conducted to evaluate the pharmacological profiles of MC3R/MC4R in the presence of MRAPs in various vertebrates, such as human , 41, mouAgrp transgenic or Pomc, Mc3r/Mc4r deficient rodents, suggesting their essential role in body weight regulation and energy metabolism  serves as a fascinating tetrapod model for the developmental, evolutionary, and regeneration studies. One of the striking features for the axolotl is its enormous genome (~32 Gb), which consists of 14 chromosomes with large number of repetitive elements. The high percentage of repetitive sequences in the axolotl genome makes the genome assembly and gene annotation extremely difficult. Therefore, tremendous efforts have been made to construct a high-quality reference genome for the axolotl , and the protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of Tongji University.Animal procedures performed in this study were in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International . Hydrochloric acid and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd . The antibodies used in this study included Rabbit anti-Flag , Mouse anti-HA , Mouse anti-Flag , Goat anti-Mouse IgG (horseradish peroxidase (HRP)-conjugated) , and Goat Anti-Rabbit Alexa-Fluor 594 (Abcam). All primers used for full-length gene amplification, reverse transcription PCR (RT-PCR), and real-time quantitative PCR are listed in All the fragments were ligated into pcDNA3.1(+) plasmid, and the constructed plasmids were then verified by DNA sequencing. The pCre-luc  was kindly gifted by Xin Xie’s Lab of Tongji University. Human α-melanocyte stimulating hormone (α-MSH) and human adrenocorticotropin ACTH (1–24) was synthesized by Genescript . We purchased the paraformaldehyde (4%), phosphate buffered saline (PBS), bovine serum albumin, non-fat milk powder and β-mercaptoethanol from Sangon Biotech . TRNzol Universal Reagent and FastQuant RT Kit (with gDNase) were obtained from Tiangen Biotech . Tetramethylbenzidine (TMB) chromogen solution was purchased from Beyotimemc3r, mc4r, and mrap2 gene sequences, we searched the axolotl RNA-Seq dataset with amino acid sequences of the corresponding protein sequences from human (Homo sapiens), mouse (Mus musculus), cattle (Bos taurus), chicken , common lizard (Zootoca vivipara), painted turtle (Chrysemys picta bellii), clawed frog , and zebrafish (Danio rerio) as queries using TBLASTN. We then eliminated the duplicates from the initial sequence pool by using Clustal Omega . The resulting unique set of sequences was used for primer design to clone the full-length sequences of these genes. The mc3r and mc4r were amplified from genomic DNA extracted from axolotl hypothalamus. The mrap2 was amplified from cDNA of the axolotl brain tissue. The functional domain characteristics and transmembrane helices of mc3r, mc4r, and mrap2 were predicted by NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).To identify the axolotl http://www.ensembl.org/) or NCBI database (http://www.ncbi.nlm.nih.gov/). The details of all used species and sequence information are listed in mc3r, mc4r, and mrap2 among various species.The protein sequences of MC3Rs, MC4Rs, and MRAP2s of various different species, namely, Human, Macaque, Sheep, Cattle, Mouse, Chicken, Snake, Common lizard, Painted turtle, Clawed Frog, Rainbow trout, Zebrafish, and Elephant shark were obtained from ENSEMBL genome browser  from a axolotl cross-tissue transcriptome study  from a female adult axolotl were collected, and their total RNA were extracted with TRNzol Universal reagent. The reverse transcription was conducted with 0.5 or 1 μg total RNA from each tissue using FastQuant RT Kit (with gDNase). The expression levels of me study .2 incubator at 37°C. Transfection was carried out with Polyethyleneimine (PEI) Transfection Reagent  according to the manufacturer’s protocol. Empty pcDNA3.1(+) vector were added to maintain the constant total plasmid amounts for all transfections groups.Human Embryonic Kidney HEK293T cells were cultured in Dulbecco’s modified Eagles medium (DMEM) medium with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin. Cells were grown in a humidified 5% CO5 cells/ml were plated into 24-well plates before transfection. The plasmids of axolotl MC3R or MC4R and MRAP2  were transiently co-transfected into HEK293T cells along with constant amounts of pCRE-Luc  to monitor the cAMP production, and pRL-TK reporter vectors  to normalize the transfection efficiency. Empty vectors were added to each group for maintaining an identical total transfection amount. After 24 h, cell culture medium was replaced with human α-MSH/ACTH (1–24) including indicated concentration in DMEM with 0.1% BSA and incubated for 4 h at 37°C/5% CO2, while negative controls were applied by using zero peptide concentration of α-MSH/ACTH (1–24). Luminescent assay was conducted with Dual-Glo Luciferase Assay System  according to the manufacturer’s protocol. Evaluation of firefly and Renilla luminescence were performed with Spectramax ID3 multimode microplate reader . The Firefly luciferase activities of HEK293T cells expressing axolotl MC3R/MC4R were further normalized to Renilla luciferase activities.HEK293T cells at a density of 1 × 10To examine the effect of axolotl MRAP2 on the cell surface expression of MC3R and MC4R, surface epitope detection by ELISA was conducted in HEK293T cells. In brief, HEK293T cells seeded in a 24-well plate coated with 0.01% poly-D-lysine were transiently transfected with N-terminally 3×HA receptors plasmids with or without different ratios of 2×Flag-MRAP2 plasmids . After 24 h transfection, cells were fixed with 4% paraformaldehyde and then washed three times with D-PBS for 5 min each time. Next, cells were incubated for 45 min with blocking buffer (5% milk in D-PBS), followed by incubation with mouse anti-HA antibodies  for 2 h. After incubation, cells were washed again and incubated for 2 h with HRP-conjugated Goat anti Mouse IgG antibodies . Finally, after washing these cells with D-PBS for three times and incubating with TMB for 30 min, the reaction was terminated by sulfuric acid (2 mol/L). We measured the absorbance value at 450 nm using Spectramax ID3 multimode microplate reader.To detect the interaction between axolotl MC3R/MC4R with MRAP2, HEK293T cells were grown on 0.01% poly-D-lysine coated cover glasses and 2Flag-MC3R/MC4R-VF1 (Venus Fragment 1) and 2Flag-MRAP2-VF2 (Venus Fragment 2) were transfected. 2Flag-MRAP2-VF1 and 2Flag-MRAP2-VF2 were transfected simultaneously as controls. After 24 h of transfection, we removed the culture medium, washed the cells with PBS, and fixed them with 4% paraformaldehyde solution for 15 min at room temperature. Cells washed by PBS were permeabilized, and non-specific binding sites were blocked in PBS containing 5% (w/v) milk for 1 h at room temperature. Permeabilized cells were then incubated overnight with 1:2,000 rabbit anti Flag monoclonal antibodies in PBS with 5% (w/v) milk at room temperature for 2 h and then the cells were washed by PBST (PBS supplement with 0.1% tween-20) 3 times for 5 min each. Then Goat anti-rabbit Alexa-Fluor 594 secondary antibodies  were added and incubated for 2 h in the dark. Finally, the coverslips were mounted with antifade reagent with DAPI  on microscope slides. Confocal microscopy imaging was conducted with a ×63 oil objective on a Zeiss confocal microscope, and the micrographs displayed in each group were exposed and identically processed.To explore the protein interaction between the axolotl MC3R/MC4R and MRAP2, the N-terminally Flag tagged MRAP2 (2xFlag-MRAP2) and N-terminally 3×HA tagged receptors expression plasmids were constructed by cloning the coding sequence into the pcDNA3.1(+) vector. The tagged receptors and MRAP2 expression plasmids were then co-transfected into HEK293T cells and incubated for 36 h. The transfected cells were then lysed by lysis buffer with 0.75% TritonX-100 . The co-IP experiment was conducted with supernatants centrifuged from the cell lysates by overnight incubating the samples with rabbit anti HA antibody at 4°C. Protein A/G Agarose beads  were added to the cell lysates right after the overnight incubation and rotated at 4°C for 3–4 h. Beads were then washed three times in lysis buffer and centrifuged, re-suspended in SDS loading buffer. After boiling for 15 min, proteins separated on sodium dodecyl sulfate-polyacrylamide gel  electrophoresis along with the mouse anti-HA antibodies for receptor proteins and mouse anti-Flag antibodies for MRAP2 proteins were used to perform western blot.mc3r, mc4r, mrap2, pomc, and agrp after three-week fasting in the axolotl, 6 juvenile axolotls were used for the food deprivation experiment with 3 as a control group and 3 in the experimental group. All juvenile axolotls from both groups were kept at the room temperature with sufficient oxygen supplements, and water in each tank was changed every day. The animals in the control group were fed twice a day for 3 weeks, whereas the animals in the experimental group were fasted for 3 weeks without any food. The body weight for each axolotl was measured before and post the experiment. The hypothalamic tissues from both control and fasting group were dissected and stored in TRNzol. The total RNA extraction and first-strand reverse transcription were conducted with TRNzol Universal reagent and FastQuant RT Kit (with gDNase) following the standard protocols. We then diluted the cDNA reaction to 1:10 as a template for qPCR. The qRT-PCR was performed using SuperReal PreMix Plus (SYBR Green) with the PCR setting as following: preincubation at 95°C for 600 s; 45 cycles of 95°C 10 s, 60°C for 10 s, and 72°C for 10 s; melting at 95°C for 10 s, 65°C for 60 s and 97°C for 1 s. All reactions were run in three biological replicates for both control and fasting groups and repeated three times.To evaluate the metabolic expression changes of 50) values was calculated using GraphPad Prism 6 . The receptor surface expression was also analyzed using GraphPad Prism 6. We applied one-way ANOVA with Tukey post-test with a nominal significant level of 0.05 (*p <0.05). The final results were represented as mean ± SEM.To validate the results of all experiments, we separately repeated these experiments at least three times. The dose–response curves for cAMP luminescent assay were analyzed with the nonlinear regression models, and the evaluation of the corresponding half-maximal effective concentration  and melanocortin accessory protein (mrap2) in the axolotl genome  regions like other GPCRs, while MRAP2 includes one TM domain like MRAPs in other species.Using multiple blast tools from the NCBI database, we identified the full-length gene sequences of the melanocortin receptors , ruminant , rodent (Mouse), avian (Chicken), reptile , amphibia , teleost , and cartilaginous fish (Elephant shark), as shown in To reveal the phylogenetic relationship of these melanocortin genes, the ML phylogenetic trees were constructed with the protein sequences of the representative vertebrates. The consensus phylogenetic trees of MC3R, MC4R, and MRAP2 are shown in mc3r, mc4r, and mrap2 genes across species, including human, mouse, chicken, turtle, clawed frog, zebrafish, and axolotl. We found that all flanking genes of axolotl MC3R shared flanking genes in other species except for diaph1 and bmp7  in zebrafish. The adjacent genes of two mrap2 copies in zebrafish were hardly overlapped with other species, suggesting a unique evolutionary pattern in teleost.Synteny analysis was performed to provide additional aspects for the homology of mc3r, mc4r, mrap2, agrp, and pomc in axolotl, quantitative RT-PCR analysis was performed with the cDNA libraries from 16 tissues of a female adult axolotl  to characterize the pharmacological profiles of axolotl MC3R and MC4R t manner Table 1.in vitro, we examined different transient transfection ratios of MCRs to MRAP2 . Noticeably, the stimulation of MC3R was dramatically suppressed by MRAP2 . The axolotl MRAP2 consists of 216 amino acids but is less conserved comparing to the corresponding genes of other species (average identity of 59%). The phylogenetic analysis indicates that both axolotl MC3R and MC4R are closely grouped with the corresponding counterparts in the clawed frog, suggesting the potential functional similarity of these MCRs among these species. We next compared the flanking genes of mc3r and mc4r across 7 different species and found the flanking region of mc3r was more conserved among these species than that of mc4r. Syntenic blocks of mc3r and mc4r in the zebrafish varied evidently from other species, indicating an early evolutionary separation of these genes in teleosts. The phylogenetic analysis of MRAP2 revealed a less conserved relationship among species, and axolotl MRAP2 fell in between the reptile and amphibian clade. A unique feature of two copies of mrap2 existent in the zebrafish and rainbow trout genome, suggests that there could be a teleost-specific duplication of mrap2 genes in the teleosts. The synteny analysis of mrap2 revealed that the flanking region of mrap2 was generally conserved among species, except for that in the zebrafish.In this study, obtaining the full-length sequences of axolotl mc3r, mc4r, and mrap2 are identified in many species. In human, MC3R is enriched in the brain region and has a very low abundance in other peripheral tissues such as testis and adrenal gland. Human MC4R exhibits high expression level in the brain, fallopian tube, and retina, moderate abundance in thalamus, olfactory bulb, and spinal cord, but low abundance in salivary gland, adrenal gland, spleen, pituitary, stomach, testis, and ovary. In addition to high expression level of human brain, MRAP2 shows a broader distribution among other human tissues than MC3R/MC4R, indicating that its function might not limit to the energy homeostasis. In the clawed frog, mc3r and mc4r are expressed in brain region with low abundance in some peripheral tissues, whereas mrap2 distributes widely among multiple tissues including brain  can be found in the article/The animal study was reviewed and approved by the Tongji University.XW, GL, JG, and ChZ participated in the design of the study. XW, SX, and XiL performed the experiments. LL, CoZ, WS, XL, YF, YL and HZ contributed to data collection and data analysis. XW, GL, and ChZ contributed to the manuscript writing. All authors contributed to the article and approved the submitted version.The work was supported by grants from the National Key Research and Development Program of China (Grant Nos. 2019YFA0111400 and 2017YFA0103902); the National Natural Science Foundation of China ; the Innovative Research Team of High-level Local Universities in Shanghai (Grant No. SSMU-ZDCX20180700) and a Key Laboratory Program of the Education Commission of Shanghai Municipality (Grant No. ZDSYS14005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gastric cancer (GC) is one of the major malignancies worldwide. Emerging evidence has revealed the potential involvement of long noncoding RNA (lncRNA) in human genetic disorders and cancer, but the role of LOC100505817 remains unknown. Thus, in this study, we isolated tissues from GC patients to characterize the functional importance of LOC100505817 in GC tumorigenesis. We also proposed a hypothesis that the regulation of Wnt/β-catenin pathway by LOC100505817 was regulated by miR-20a-mediated WT1. After the collection of cancer tissues and adjacent tissues were obtained from GC patients, expression of LOC100505817, Wnt/β-catenin pathway- and EMT-related genes was quantified. Ectopic expression and knockdown experiments were applied in order to investigate the protective role of LOC100505817 in the progression of GC. Subsequently, cell viability, flow cytometry for apoptosis and cell cycle were detected via CCK-8, while migration and invasion were determined using scratch test and Transwell assay respectively. Then interactions among LOC100505817, miR-20a and WT1 were explored by dual luciferase reporter gene assay, RNA pull down assay and RNA binding protein immunoprecipitation (RIP) assay. The results found poor expression LOC100505817 was poorly expressed in GC cells and tissues. Overexpressed LOC100505817 resulted in the significant reduction of cell proliferation, migration and invasion as well as the expression of Wnt2b, β-catenin, CyclinD1, N-cadherin, Vimentin and snail, while increased cell apoptosis along with the expression of E-cadherin. Wnt/β-catenin pathway and EMT in GC cells were suppressed by LOC100505817 through miR-20a-inhibted WT1. In summary, our results provided evidence suggesting that LOC100505817 inhibits GC through LOC100505817-mediated inhibition of Wnt/β-catenin pathway, that leads to the overall restraining of GC cell proliferation, migration and invasion through miR-20a-reduced WT1. The probe ID was converted to genesymbol by using the annotation information, Then, with the normal sample serving as the control, an empirical Bayes method was conducted to screen out differentially expressed genes (DEGs) based on “limma” package of the Bioconductor using R language  ethanesulfonic acid (HEPES) at the ratio of 1:2:50. With 1 × 10t test, and multi-group comparisons were carried out by one-way analysis of variance (ANOVA). Comparisons among multiple groups at different groups were analyzed by two-way ANOVA, followed by Bonferroni post-hoc test. p < 0.05 was indicative of significant difference.All data were processed using SPSS 21.0 software . The measurement data was depicted by mean ± standard deviation. Comparisons between GC tissues and adjacent tissues were performed by p < 0.05). Besides, western blot analysis (p < 0.05).By analyzing the GSE13911 microarray, we found a low expression the LOC100505817 was lowly expressed in GC . In ordeanalysis , illustrp < 0.05). A significant decrease of LOC100505817 expression was observed in patients with tumor diameter ≥5, LNM, or TNM at III – IV stage when compared with patients with tumor diameter <5, or without LNM, or TNM at I – II stage . There was no significant difference between LOC100505817 and age, sex and tumor differentiation. These findings evidently revealed that LOC100505817 could potentially be a factor involved in the progression of GC.Next, with the attempt to determine the involvement of LOC100505817 in the progression of GC, the correlation between the LOC100505817 expression and clinical features of the enrolled patients with GC was assessed. As shown in LOC100505817 expression was detected in five different GC cell lines  by RT-qPCR. The expression of LOC100505817 in BGC823 cells was 3.9 ± 0.14, in AGS cells was 2.75 ± 0.09, in MKN45 cells was 1.1 ± 0.11, in SGC7901 cells was 6.0 ± 0.18 and in MKN28 cells was 3.2 ± 0.15 . The expp < 0.05), and the si-β-catenin group presented with the opposite results.Next, the correlation of LOC100505817 and Wnt/β-catenin with EMT process was further detected in GC cells. First, LOC100505817 and β-catenin were silenced in SGC7901 cells by siRNA. RT-qPCR results revealed that after siRNA treatment, LOC100505817 and β-catenin expression was decreased in SGC7901 cells . RT-qPCRin vitro by Transwell assay. The results demonstrated that compared with the si-NC groups, the migration ability of SGC7901 cells in si-LOC100505817 group was significantly increased, while that of SGC7901 cells were significantly reduced in the si-β-catenin group . The growth rate of SGC7901 was markedly accelerated in the si-LOC100505817 group, and the OD value at the 48th and 72nd h was significantly higher than that in the blank and si-NC groups . The growth rate of SGC7901 obviously reduced in the pGMLV-LOC100505817 group, in which the OD value at the 48th and 72nd h was significantly higher than that in the blank and pGMLV-vector groups . Besides, it was also observed that relative to the pGMLV-LOC100505817 group, the inhibited cell proliferation was reversed in the pGMLV-LOC100505817 + oe-β-catenin group . These data suggest that LOC100505817 has an inhibitory effect on GC SGC7901 cell proliferation through downregulating β-catenin.Subsequently, we assessed the roles of LOC100505817 on GC cell proliferation. First, the efficiency of lentivirus transfection and β-catenin mRNA expression were detected by RT-qPCR. The results showed that the LOC100505817 expression was evident lower and the mRNA expression of β-catenin was obvious higher in the si-LOC100505817 group than that in the si-NC group, while the LOC100505817 expression was obvious higher and the mRNA expression of β-catenin was significant lower in the pGMLV-LOC100505817 group than that in the pGMLV-vector group (both p > 0.05). Cell migration in the si-LOC100505817 group was notably promoted, but significantly weakened in the pGMLV-LOC100505817 group versus that in the blank group (both p < 0.05). Moreover, as compared with the pGMLV-LOC100505817 group, the inhibited cell migratory capability was reversed in the pGMLV-LOC100505817 + oe-β-catenin group . It indicates that GC SGC7901 cell migration is inhibited by LOC100505817 overexpression.Next, in order to determine GC cell migration and invasion influenced by LOC100505817, starch test and Transwell assay were used. Results of scratch test ,B showedp > 0.05). Cell invasion in the si-LOC100505817 group was remarkably enhanced, but significantly repressed in the pGMLV-LOC100505817 group relative to that in the blank group . In addition, the inhibited cell invasive capability was reversed in the pGMLV-LOC100505817 + oe-β-catenin group than in the pGMLV-LOC100505817 group   . These rp > 0.05). In comparison with the blank and si-NC groups, cell number in G1 phase was decreased in the si-LOC100505817 group yet increased in S phase . The pGMLV-LOC100505817 group was observed to have increased number of cells in the G1 phase and decreased cells in the S phase . Compared with the pGMLV-LOC100505817 group, the pGMLV-LOC100505817 + oe-β-catenin group showed decreased cell number in G1 phase and increased cell number in S phase .Furthermore, for the purpose to detect the functions of LOC100505817 on GC cell apoptosis and cell cycle, flow cytometry was employed. PI single staining results  showed tp > 0.05). Apoptosis rate was reduced significantly in the si-LOC100505817 group while notably enhanced in the pGMLV-LOC100505817 group versus that in the blank and si-NC groups . Moreover, relative to the pGMLV-LOC100505817 group, the cell apoptosis was observed to decrease in the pGMLV-LOC100505817 + oe-β-catenin group . These results suggest that overexpressed LOC100505817 can accelerate GC SGC7901 cell apoptosis and arrest GC SGC7901 cell at G1 phase.The results obtained from Annexin V/PI double staining  confirmep > 0.05). In comparison with the blank group, expression of Wnt2b, β-catenin, CyclinD1, Vimentin, N-cadherin and snail was remarkably up-regulated while that of LOC100505817 and E-cadherin was notably diminished in the si-LOC100505817 group . Contrary to that, a profound decline was shown in the expression of Wnt2b, β-catenin, CyclinD1, Vimentin, N-cadherin and snail in the pGMLV-LOC100505817 group, with a dramatical enhancement in expression of LOC100505817 and E-cadherin . When compared with the pGMLV-LOC100505817 group, the expression of Wnt2b, β-catenin, CyclinD1, Vimentin, N-cadherin and snail was elevated in the pGMLV-LOC100505817 + oe-β-catenin group, while expression of E-cadherin was significantly reduced . Therefore, LOC100505817 might potentially contribute to the suppression of the Wnt/β-catenin signaling pathway and EMT.Finally, with the attempt to explore the impacts of LOC100505817 on Wnt/β-catenin signaling pathway and EMT, RT-qPCR and western blot analysis were performed to detect expression of LOC100505817, Wnt2b, β-catenin, CyclinD1, Vimentin, N-cadherin, E-cadherin as well as snail. As displayed in Gastric cancer has a high incidence and mortality worldwide , 28. AccWe initially demonstrated that elevation of LOC100505817 expression resulted in the significant suppression of GC progression through the inhibition of the proliferation, migration and invasion while inducing apoptosis of GC SGC7901 cells. LncRNA are recognized as novel regulators in the cancer paradigm and may possess potential values in oncogenic as well as tumor suppressive pathways and a wide range of human tumors have been found to have dysregulated lncRNA . A priorOur data also indicated that the expression of relevant genes linking to Wnt/β-catenin signaling pathway and EMT was elevated secondary to LOC100505817 silencing. A previous study also suggested that the Wnt-1, Wnt-2 and Wnt-2b, proteins in the Wnt family, are up-regulated in GC, and the up-regulation of Wnt-2 was positively correlated to an increase in metastatic potential . Wnt/β-cCollectively, the findings obtained in this study summarized that human GC cells have a low expression of LOC100505817. Ectopic expression of LOC100505817 correlated with cellular process of GC. These results further highlight the need to focus on LOC100505817 as a potential biomarker for GC. However, additional studies are recommended in order to lay a theoretical foundation for better prevention, diagnosis, and treatment measures on a genetic level.


Murraya koenigii (Rutaceae) is utilized in Ayurvedic medicine for various ailments. Pharmacological studies evidenced its potential antioxidant, anti-inflammatory, anticancer, hepatoprotective, immunomodulatory, antimicrobial, and neuroprotective activities, among others. In line with our interest in exploring natural agents for the treatment of neurodegenerative diseases, this review presents an overview of literature concerning the mechanisms of action and the safety profile of significant bioactive components present in M. koenigii leaves to support further investigations into their neuroprotective therapeutic potential.Neurodegenerative diseases (NDs) mainly affect neurons and gradually lead to a loss of normal motor and cognitive functions. Atypical protein homeostasis—misfolding, aggregations and accumulations, oxidative stress, inflammation, and apoptosis—are common features in most NDs. To date, due to the complex etiology and pathogenesis of NDs, no defined treatment is available. There has been increasing interest in plant extracts as potential alternative medicines as the presence of various active components may exert synergistic and multi-pharmacological effects.  Neurodegenerative diseases (NDs) are a broad term used to define a range of disorders  primarily affecting the neurons and leading, ultimately, to the progressive loss of normal motor functions and the decline in cognitive functions. The brain region affected, severity, and pace of neurodegeneration vary with the type of ND. According to a recent report, around 50 million people worldwide have such diseases . The totThe exact reasons for ND manifestation among some people and not for others are still not fully understood. Some NDs are suspected to occur due to genetic mutations; some are also linked to living conditions . HoweverMurraya koenigii (curry-leaf tree) was a small, tropical to sub-tropical shrub or tree growing up to 6 m in height, belonging to the family Rutaceae. It can be found in countries like India, Sri Lanka, and Bangladesh. Almost every part  of this plant is used in Indian traditional system of medicine (Ayurveda) to treat various ailments. The green curry leaves were generally used in treating piles, inflammation, itching, fresh cuts, dysentery, and edema. The roots were used for curing body aches while the bark was helpful in treating snakebites [M. koenigii leaves are known for their signature aroma, slightly spicy and bitter taste; these characteristics are preserved even after drying. Fresh and dried curry leaves are considered as an important ingredient in South Indian cuisine and are broadly used for seasoning and flavoring dishes. The curry leaves have well established therapeutic potentials like hypoglycemic, hypolipidemic [akebites . Fresh Mipidemic ,13,14,15ipidemic ,17, hepaipidemic ,19, gastipidemic , cardiopipidemic ,22, atheipidemic  and cholipidemic . The alcipidemic ,26, analipidemic ,28,29, aipidemic , antitriipidemic , antibacipidemic ,33, antiipidemic , antileiipidemic , antidiaipidemic , wound-hipidemic , anti-obipidemic , anticanipidemic ,40,41, aipidemic ,43. The ipidemic ,45,46 byipidemic ,48 and dipidemic . M. koenigii leaf extract and its bioactive components that exhibit neuroprotective effects through various mechanisms for the prevention and treatment in NDs. The literature search was conducted using Google Scholar and PubMed online databases until November 2021 with key words being neuroprotective, Murraya koenigii leaf, chemical composition and essential oils. Additionally, the safety evaluation of M. koenigii leaves is explored.This review is focused on the M. koenigii revealed the following contents: ash (15.60% ± 0.21), moisture (23.40% ± 0.10), protein (8.38% ± 0.02), fats (6.48% ± 0.22), carbohydrate (39.44% ± 0.04), and crude fiber (6.30% ± 0.05) [M. koenigii are also a rich source of vitamins and minerals. The compositional analysis (in mg per 100 g) revealed ascorbic acid , β-carotene , niacin , riboflavin , thiamin , and α-tocopherol  [M. koenigii revealed moisture content (64.9%), total sugar (9.76%), reducing sugar (9.58%), ascorbic acid (13.35%), calcium (0.811%), iron (0.007%), magnesium (0.166%), phosphorous (1.97%), and potassium (0.082%) [Proximate analysis on the leaves of  ± 0.05) . Phytoch ± 0.05) . The lea ± 0.01) . Among t ± 0.01) . Interes(0.082%) . M. koenigii, including the leaves, roots, stem bark, fruits, and seeds, using chromatographic and spectroscopic techniques. The isolation of terpenoids, alkaloids, flavonoids, coumarins, polyphenols, and essential oils was described in phytochemical studies searching for potential biologically active constituents [M. koenigii was reported as a rich source of more than fifty carbazole-type alkaloids with potent pharmacological activities [M. koenigii included mukoeic acid, murrayanine, mukonicine, koenigine, koenine, 9-formyl-3-methylcarbazole, murrayanol, and O-methylmurrayamine A, while additional antimicrobial alkaloids were girinimbine, murrayanine, mahanine, murrayacine, mukoline, and pyrafolione D [M. koenigii, the coumarins and flavonoids of catechin, epicatechin, quercetin, myricetin, naringin, and rutin were identified as antioxidant and anticancer compounds. Additionally, the polyphenolic compounds ferulic acid, gallic acid, and vanillic acid were reported to increase the antioxidant potential of M. koenigii [Many structurally diverse natural products were identified from tituents ,53. In ptivities . Cytotoxtivities ,54. Mahativities . Other rolione D ,54. AmonM. koenigii leaves from different regions [M. koenigii were allo-ocimene, α-terpinene, (E)-β-ocimene, elemol, linalool, geranyl acetate, and myrcene [M. koenigii leaves collected from Tamilnadu, India, were extracted by hydrodistillation [E)-β-ocimene (3.68%), elemol (7.44%), geranyl acetate (6.18%), linalool (32.83%), myrcene (6.12%), α-terpinene (4.9%), and neryl acetate (3.45%) were present in the highest amounts [trans-β-caryophyllene , β-caryophyllene , and bicyclogermacrene  were identified as the major constituents [M. koenigii essential oils from the London, United Kingdom were β-caryophyllene (28.7%), β-gurjunene (21.4%), selinene (8.2%), β-elemene (6.8%); essential oil from Nigeria contained oxygenated compounds including β-caryophyllene (20.5%), bicyclogermacrene (9.9%), α-cardinol (7.3%), caryophyllene epoxide (6.4%), and β-selinene (6.2%); and samples from Bangladesh contained 3-carene (54.2%) and caryophyllene (9.5%) [M. koenigii collected from Dehra Dun, India, were hydrodistilled and subjected to GC–MS analysis. Among the 34 compounds identified, major constituents were α-pinene (51.7%), sabinene (10.5%), β-pinene (9.8%), β-caryophyllene (5.5%), limonene (5.4%), bornyl acetate (1.8%), terpinen-4-ol (1.3%), γ-terpinene (1.2%), and α-humulene (1.2%) [M. koenigii fresh leaves were collected from six geographical locations in Malaysia and the essential oils were obtained by hydrodistillation [M. koenigii depending on the geographical locations and conditions including soil type and climate [Over 100 compounds were identified in the essential oils of  regions ,33,55,56 myrcene . M. koenillation . Gas chr amounts . Leaf sa climate .M. koenigii leaf extracts and specified metabolites against their potential targets in NDs. The oral administration of the leaf aqueous extract  to aluminum-treated rats was found to significantly restore antioxidant parameters , reversing them to normal through the neuroprotective properties of the extract [M. koenigii on oxidative stress was most likely due to carbazole alkaloids, polyphenols, and flavonoids, which are known to have antioxidant properties [M. koenigii leaves methanolic extract [M. koenigii leaves (MKL). MKL (leaf powder mixed with wheat flour) were orally administered to various groups, and their behaviors were evaluated with the elevated plus-maze and Hebb–Williams maze trials. A significant dose-dependent improvement in the memory scores of young and aged rats was reported, and the significant reduction in amnesia could be due to the induction by scopolamine  and diazepam  in the treatment groups [M. koenigii alkaloid extract (MKA) caused a dose-dependent reduction in AChE levels in the brain of aged mice. It also prevented the brain from age-induced oxidative stress by decreasing lipid peroxidation and nitric oxide levels and increasing the level of antioxidant enzymes  in the brain [This section describes the neuroprotective effects of  extract . The prooperties ,59,60. Noperties . The pos extract . Diazepat groups . In addit groups . Furtherhe brain ,66. 50 4 µg/mL), which was responsible for production of β amyloid (Aβ). In the in vivo study, mahanimbine supplementation re-equilibrated the levels of antioxidant markers  in the brain in comparison to the untreated group [50 0.03 mg/mL) of mahanimbine was also established in vitro under acellular conditions [The neuroprotective potential of the carbazole alkaloid mahanimbine against lipopolysaccharides (LPS)-induced neuronal deficits in the SK-N-SH neuroblastoma cell line and its antioxidant potentials in the ICR mouse brain were evaluated. The pre-treatment of SK-N-SH cells with mahanimbine significantly mitigated the generation of LPS-induced reactive oxygen species (ROS) in vitro. In addition, mahanimbine also inhibited the β-secretase , 2,2-azino-bis , hydroxyl radical, nitric oxide, hydrogen peroxide, super oxide radical scavenging, ferric reducing antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP) and reducing power assays, respectively [A tricyclic sesquiterpene, isolongifolene (ILF), present in the essential oil of in vitro  and in vin vitro . ILF disectively . ILF attectively . AKT regectively . The actectively . These eectively , which iectively , ILF impectively . M. koenigii leaves [A sesquiterpene alkene and atypical dietary cannabinoid, β-caryophyllene (BCP), is another essential bioactive compound in i leaves . It is ai leaves , and is i leaves ,81. Throi leaves ,83. In ti leaves . Similari leaves . In AD, i leaves ,87,88. Ii leaves . Hence, i leaves . The effect of BCP on cognition was studied in a galactose (GAL) model of aging in mice. GAL induction lead to increased DNA oxidation in the prefrontal cortex and increased astrocyte numbers and interactions in the hippocampal region of the brain. Although the cognitive damage was not reversed by administering BCP, it was able to block the increases in the number of astrocytes and DNA oxidation in mice treated with GAL. Hence, the neuroprotective effects of BCP were demonstrated at the molecular and cellular level in the GAL model of aging . The proM. koenigii [Spathulenol , a tricyclic sesquiterpenoid, is a component of essential oil from the leaves of koenigii . Spathulkoenigii . M. koenigii leaf [50 0.2 mM) [50 5.3 µM) and geranyl acetate (IC50 244.5 μM) [M. koenigii leaf [(+)-3-Carene, a bicyclic monoterpenoid from gii leaf , was rep 0.2 mM) . A molec44.5 μM) , as compgii leaf ,78. M. koenigii leaves. The improved cognitive-enhancing effects and anti-apoptotic activities in Aβ1-42-treated rats could mostly result from its antioxidant potential [Linalool, a monoterpene alcohol, was also present in the essential oil of otential . In anototential . Linaloootential . In addiotential ,108. Morotential .M. koenigii, were studied. MYR treatment protected the C57BL/J6 mice against oxidative stress, apoptosis, and histopathological damage induced by cerebral ischemia/reperfusion (I/R) [The neuroprotective effects of β-myrcene (MYR), a monoterpene present in the leaves of on (I/R) . M. koenigii leaves from the studies with a rotenone-induced model of PD [The neuroprotective effect of nerolidol (NRD), a sesquiterpene alcohol, was discovered in the essential oil of el of PD . NRD supel of PD . In anotel of PD . TherefoM. koenigii against various NDs are shown in M. koenigii extracts and natural products are shown in M. koenigii and chlorhexidine gluconate for the treatment of gingivitis [M. koenigii, the research was lacking on its preclinical and/or clinical efficacy. Therefore, there is an urgent need to conduct clinical trials to prove the neuroprotective capacity of M. koenigii.Currently, no significant clinical trials have been conducted on curry leaves. A randomized controlled clinical trial was conducted to evaluate the efficacy of ngivitis . In anotngivitis . An herbngivitis . The effngivitis . HoweverM. koenigii were collected from different regions, and their elemental analyses were performed by the instrumental neutron activation (INAA) and atomic absorption spectrophotometry (AAS). Mean elemental contents varied owing to different climate, soil, and other geo-environmental conditions. The changes in plants grown on polluted soils revealed the higher levels of heavy metals, which posed a major health concern, where they could affect kidney, liver, and the central nervous system [Curry leaves were rich in calcium, potassium, magnesium, and phosphorous, along with trace amounts of zinc, manganese, selenium, and iron. Additionally, lead, mercury, and cadmium were also present, but below the US FDA limits . Variouss system .M. koenigii leaf (300 and 500 mg/kg) for 28 days. In addition, no mortality was observed at higher doses (900 mg/kg), but congestion, hemorrhage, and lymphocyte infiltration were recorded. The study concluded that the safety consumption could be high as 500 mg/kg dosage without inducing any structural damage to the organs [50 = 200 mg/kg/day was reported [50 = 316.23 mg/kg body weight) to rats and to cause liver inflammation. However, this dose had no severe side effects on other organs [No signs of mortality or morbidity were seen in either male or female rats fed the ethanolic extract of e organs . Similarreported . In addireported . Anotherreported  However,M. koenigii leaves in ameliorating several neurodegenerative disorders. M. koenigii leaves are an integral part of Indian cuisine and are used for various ailments traditionally. Carbazole alkaloids are abundantly found in species of the Rutaceae family including M. koenigii (Curry leaves). Both the natural and synthetic derivatives of carbazole alkaloids revealed numerous pharmacological activities, including neuroprotection. The M. koenigii leaf extracts and their bioactive compounds, including carbazole alkaloids, sesquiterpenoids, and monoterpenoids, exhibited a multi-target approach by alleviating the oxidative stress, attenuating proinflammatory cytokines, inhibiting AChE and BACE 1, preventing/reducing Aβ protein aggregation, and improving cognitive dysfunction. Thus, M. koenigii leaves, their extracts, or purified compounds could offer a beneficial alternative therapy to treat NDs through their multi-targeted neuroprotective properties and by improving cholinergic transmissions. Hence, the bioactive compounds from the leaves can serve as lead molecules in future drug discovery. Moreover, there have been no safety concerns associated with the use of M. koenigii leaves. Therefore, it is highly recommended to determine effective dose of leaves or their compounds in human for future clinical trials on NDs.Traditional medicinal plants have gained importance during the last few decades, and evidence has been presented for their therapeutic actions, allowing the traditional knowledge of plants to be streamlined with the modern system of medicine to achieve health benefits. This review presented substantial evidence in relation to the mechanistic aspects of


Background: Pharmacological research results showed that total flavonoids of Chuju (TFCJ) could be used to treat acute myocardial ischemia and myocardial ischemia-reperfusion injury. In this study, we explored the protective effect of TFCJ on ischemic stroke (IS) in the IS rat model. We hypothesized that TFCJ might exert its neuroprotective effects by suppressing apoptosis and oxidative stress that are closely related to PI3K/Akt/mTOR signaling pathway.Method: TFCJ  was administered for 7 days. Rats (260 ± 20 g) were subjected to middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 24 h. The neuroprotective effect of TFCJ was substantiated in terms of neurological deficits, oxidative stress , pathomorphological changes (HE staining and TUNEL staining), and neurobehavioral functions in the rats. Then, we employed network pharmacology to reveal the potential mechanism of TFCJ against IS. Western blot was used to determine the levels of PI3K/AKT/mTOR pathway proteins. The expression of BCL-2, BAX, and cleaved-Caspase-3 was also measured by Western blots and RT-PCR.Results: The histopathological assessment showed that TFCJ reduced MCAO-induced brain damage. Besides, TFCJ exerted a protective role in MCAO rats by alleviating cell apoptosis and oxidative stress. Network pharmacology showed that TFCJ might be used against IS through the PI3K/AKT signaling pathway. TFCJ reduced cell apoptosis and oxidative stress by increasing the level of p-AKT and p-mTOR in MCAO rats, while the effect of TFCJ was significantly reversed when applying LY294002 (PI3k inhibitor).Conclusion: These results indicated that TFCJ might decrease oxidative stress and apoptosis that are closely related to PI3K/Akt/mTOR pathway in IS. TFCJ is a promising authentic traditional Chinese medicine for the management of IS. Stroke is a common disease characterized by a decreased or blockage in the brain’s blood supply, which accounted for 11.59% of all deaths in 2019 . In the 2 +overload , mixed and sonicated 60°C for 40 min, then concentrated in a rotary evaporato as described by the previous method . The cruExperiments were conducted on 7-weeks old-male Sprague-Dawley rats (240–280 g body weight). All the animals were purchased from Huaxing Experimental Animal Farm , and maintained in standard housing conditions in colony cages 12 h light/dark cycles with free access to food and water. The animal care and experimental procedures were approved by Animal Ethics Committee of Anhui Science and Technology University. Because estrogen has a certain effect on the experimental results, male rats were selected as experimental animals.–1). We slowly inserted the threaded plug from the left external carotid artery (ECA) through the common carotid artery (CCA) into the internal carotid artery (ICA) until the origin of middle cerebral artery (MCA) was blocked as the previous literature . Sham group, MCAO group, TFCJ-H group (40 mg⋅kg–1 TFCJ), TFCJ-M group (20 mg⋅kg–1 TFCJ), TFCJ-L group (10 mg⋅kg–1 TFCJ), Xuesaitong group .To understand the relationship between TFCJ anti-IS and apoptosis and oxidative stress, 120 rats were randomly divided into six groups (n = 10/group) as follows:Sham group, MCAO group, TFCJ group (40 mg⋅kg–1 TFCJ), TFCJ + LY group .To explore the relationship between TFCJ anti-IS and PI3K/Akt/mTOR pathway, 40 rats were randomly divided into four groups  was employed for infarct area analysis. Red stained areas corresponded to unaffected areas, while that stained in white was the infarcted area . The cerThe brain hemisphere ipsilateral to MCAO was homogenized with pre-cooled physiological salt, centrifuged like the blood, and the supernatant was immediately collected and stored in the refrigerator at −80°C until use. SOD, GSH-Px, CAT activities, and the content of MDA were deteThe animals were anesthetized and decapitated, and brains were collected. The brain sample was embedded in paraffin, coronally sliced at 5-μm, and measured by HE staining , and ter1) (2) (3)  , Integra1) (2) , and Bio (2) (3) . The com) (3)  following standard techniques.Total RNA of the ischemic penumbra of brain tissue was extracted using RNA isolater Total RNA Extraction Reagent . The isolated RNA samples were reverse transcribed using HiScriptData of BCL-2, BAX, and cleaved-Caspase-3 mRNA were normalized to β-actin mRNA . RT-PCR We performed Western blot analysis following previously described protocols . The tott-tests were used for comparison of variable pairs. P < 0.05 was considered as statistically significant.All data were expressed as mean ± SD; SPSS 25.0 statistical software  was adopted for all statistical analyses. The multiple variables were performed by one-way analysis of variance (ANOVA), and student’s ##P < 0.01); the neurological deficits in the rats pre-treated with TFCJ  were significantly lower than those in the MCAO group ; the neurological deficit scores in the Xuesaitong group significantly decreased compared to the MCAO group (**P < 0.01).The neurological deficit scores and cerebral infarction area were evaluated  after re##P < 0.01). Cerebral infarct area in three dose groups of TFCJ was much lower than MCAO group (**P < 0.01). The cerebral infarct area in the Xuesaitong group was significantly decreased than the MCAO group (**P < 0.01).The sham group had a smaller cerebral infarct area, while the MCAO group had a bigger infarct area (##P < 0.01), and the concentration of MDA was higher in the MCAO group than in sham group (##P < 0.01), indicating that MCAO induced oxidative stress in the brain tissue and serum.The activity of SOD, GSH-Px, and CAT in the brain tissue and serum were lower in the MCAO group than in the sham group (**P < 0.01). The concentration of MDA in the brain tissue and serum of the three-dose groups of TFCJ was significantly lower compared to the MCAO group .The activity of SOD, GSH-Px, and CAT in the brain tissue and serum of high and middle dose groups of TFCJ was much greater than the MCAO group (**P < 0.01), and the activity of CAT in the serum and brain tissue was significantly increased (P < 0.05).Compared with the MCAO group, the activity of SOD and GSH-Px in the serum of the TFCJ-L group was significantly improved (**P < 0.01), and the concentration of MDA was significantly decreased (**P < 0.01).Compared with the MCAO group, the activity of SOD, GSH-Px, and CAT in the brain tissue and serum of the Xuesaitong group was significantly improved , while they were reduced in the TFCJ group (**P < 0.01). Compared to the MCAO group, the numbers of TUNEL-positive neurons were significantly lower in the Xuesaitong group (**P < 0.01).The nucleus was stained in blue, and the TUNEL-positive neurons were brown. Almost no neuronal death was observed in the sham group. The numbers of TUNEL-positive neurons were significantly higher in the MCAO group than in the sham group  chroman-4-one, linarin, diosmetin, chryseriol, naringenin, and artemetin in TFCJ are described in IS-related targets were identified from GeneCards, OMIM, and DisGeNET databases. After deduplication, all information were merged, resulting in a database of 5,111 IS-related targets, including 934 common targets . A totalP-value < 0.05 , 50 cellular components (CC), 108 molecular functions (MF), and 110 KEGG pathways met the screening criteria of e < 0.05 –5. The te < 0.05 .The top 20 terms of KEGG pathways were pathways in cancer, PI3K-Akt signaling pathway, Ras signaling pathway, proteoglycans in cancer, Rap1 signaling pathway, FoxO signaling pathway, insulin signaling pathway, estrogen signaling pathway, pancreatic cancer, thyroid hormone signaling pathway, prolactin signaling pathway, prostate cancer, colorectal cancer, VEGF signaling pathway, central carbon metabolism in cancer, Fc epsilon RI signaling pathway, adherens junction, chronic myeloid leukemia, PPAR signaling pathway, and non-small cell lung cancer .The first part of the article found that TFCJ has a brain-protective effect and was related to the inhibition of oxidative stress and cell apoptosis. As the top 20 of the KEGG pathway, the PI3K-Akt signaling pathway has an important role in apoptosis and oxidative stress. Therefore, we focused on the regulation of TFCJ on the PI3K-Akt signal pathway and its downstream mTOR signal pathway (the 86th position of the KEGG pathway).##P < 0.01). The p-Akt and p-mTOR expression in the TFCJ treatment rats were significantly higher than in the MCAO control rats (**P < 0.01). After using LY294002, the TFCJ treatment was reversed (see ▲▲P < 0.01). There was no significant change in total Akt and mTOR protein expression in each group (P > 0.05).The p-Akt and p-mTOR expression in the MCAO control rats were significantly lower than in the sham rats (##P < 0.01). In the TFCJ treatment animals, GSH-Px (**P < 0.01). After using LY294002, the effect of TFCJ was reversed (▲▲P < 0.01). The result showed that TFCJ could inhibit oxidative stress caused by MCAO in the serum through PI3K/Akt/mTOR pathway.Compared with the sham group, the activities of SOD , GSH-Px , GSH-Px  and CAT , GSH-Px  activiti, GSH-Px  was lowe##P < 0.01), whereas TFCJ upregulated BCL-2 protein expression (P < 0.01). The BAX and cleaved-Caspase-3 expression were increased after IS 24h (##P < 0.01), whereas TFCJ upregulated BAX and cleaved-Caspase-3 protein expression (P < 0.01). After using LY294002, the TFCJ treatment was reversed (see ▲▲P < 0.01). Moreover, RT-PCR analysis indicated that MCAO reduced the expression of BCL-2 and increased the level of BAX and cleaved-Caspase-3 (##P < 0.01), while TFCJ induced the expression of BCL-2 and decreased the level of BAX and cleaved-Caspase-3 (P < 0.01). After using LY294002, the effect of TFCJ was significantly reversed (▲▲P < 0.01).As shown in This study evaluated the neuroprotective effect of TFCJ against IS in rats. We hypothesized that TFCJ might exert its neuroprotective effects by suppressing apoptosis and oxidative stress by activating the PI3K/Akt/mTOR signaling pathway. The important predictors of IS are the neurological deficit scores and cerebral infarction . In thisNext, we explored the underlying mechanism of TFCJ against IS. Previous studies  have rep2O2 through SOD disproportionation to avoid cytotoxicity (GSH-Px is the main antioxidant that interacts with lipid hydrogen peroxide . The degtoxicity . MDA is toxicity . To asseThen, potential targets and pathways of TFCJ were screened by network pharmacology. A total of 12 compounds of TFCJ were collected. We predicted 395 targets according to their structure and screened 5111 targets of IS. Finally, 247 common targets were obtained by a Venny tool. A total of 395 biological processes, 50 cellular components, 108 molecular functions, and 110 KEGG pathways were obtained by GO and KEGG analysis of 247 common targets.PI3K/Akt/mTOR is a multifunctional signaling pathway associated with anti-apoptosis and anti-oxidant stress. Phosphorylation of Akt and mTOR were suppressed in the late stage of IS. Akt and mTOR overexpression improved the protective effects after cerebral ischemia-reperfusion injury in MCAO rats, suggesting that the activation of the PI3K/Akt/mTOR signaling pathway provides significant neuroprotective effects against cerebral ischemia-reperfusion injury. P-Akt and p-mTOR  are commTo further explore the molecular mechanisms underlying the TFCJ anti-apoptosis and anti-oxidant stress effect, we detected the expression changes of mediators of apoptosis and oxidant stress involved in ischemic brain damage under the inhibitory condition of PI3K/Akt/mTOR signaling pathway. BCL-2 is a type of anti-apoptotic protein, while BAX and cleaved-Caspase-3 are known to promote apoptosis . The proin vitro.In summary, our data suggested that TFCJ might decrease nerve damage in rat cerebral ischemia by regulating the PI3K/Akt signaling pathway. The findings of this study provide a reference for the clinical anti-apoptosis, anti-oxidant stress, and neuroprotection of TFCJ. In our future studies, we plan to further assess the effects of TFCJ on cell apoptosis and oxidative stress The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Anhui Science and Technology University institutional animal care. Written informed consent was obtained from the owners for the participation of their animals in this study.CW, HC, B-BM, and HY contributed to conception and design of the study. CW organized the database and wrote the first draft of the manuscript. CW and H-HJ performed the statistical analysis. H-HJ wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Wnt/β-catenin signaling is a highly conserved pathway related to a variety of biological processes in different cells. The regulation of replication of various viruses by Wnt/β-catenin signaling pathway has been reported. However, the interaction between the Wnt/β-catenin pathway and avian leukosis virus is unknown. In the present study, we investigated the effect of modulating the Wnt/β-catenin pathway during avian leukosis virus subgroup J (ALV-J) infection. The activation of the Wnt/β-catenin pathway by GSK-3 inhibitor increased ALV-J mRNA, viral protein expression, and virus production in CEF cells. This increase was suppressed by iCRT14, one of the specific inhibitors of the Wnt/β-catenin signaling pathway. Moreover, treatment with iCRT14 reduced virus titer and viral gene expression significantly in CEF and LMH cells in a dose-dependent manner. Inhibition Wnt/β-catenin signaling pathway by knockdown of β-catenin reduced virus proliferation in CEF cells also. Collectively, these results suggested that the status of Wnt/β-catenin signaling pathway modulated ALV-J replication. These studies extend our understanding of the role of Wnt/β-catenin signaling pathway in ALV-J replication and make a new contribution to understanding the virus–host interactions of avian leukosis virus. Alpharetrovirus of the family Retroviridae, which causes immunosuppression, reproductive disorders, and a diverse set of tumors, causing significant economic losses in the poultry industry worldwide [Avian leukosis virus (ALV) belongs to the genus orldwide ,2,3. Witorldwide . To dateorldwide . Similarorldwide  showed torldwide . HoweverThe canonical Wnt/β-catenin signaling pathway plays an important role in cell development and differentiation. It is closely associated with developmental diseases and cancer ,9,10. InSeveral reports have demonstrated the interaction of the Wnt/β-catenin signaling pathway with human and animal viruses, including human immunodeficiency virus (HIV) , herpes In this study, we investigated for the first time the relationship between the Wnt/β-catenin signaling pathway and ALV-J replication in CEF cells. Our findings provide new insights into the virus–host interaction of avian leukosis virus.2 atmosphere. LMH (ATCC® cRL2117™), a primary hepatocellular carcinoma epithelial cell line of a leghorn chicken, was maintained in F12 medium  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin at 37 °C in a 5% CO2 atmosphere. GSK-3 inhibitor X from Merck  was diluted in dimethyl sulfoxide (DMSO). iCRT14 was purchased from SIGMA  and diluted in dimethyl sulfoxide (DMSO). Lipofectamine RNAiMax and 2000 reagent were purchased from Invitrogen . The Cell Counting Kit-8 (CCK-8) for quantitation of viable cell number in proliferation and cytotoxicity assays was purchased from Vazyme Biotech company . The specific monoclonal antibodies, 5D3 and JE9, against ALV-J p27 and gp85 protein, respectively, were described in our previous reports [The JS09GY3 (GenBank ID: GU982308) strain of ALV-J was maintained at the Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University. Chicken embryo fibroblast (CEF) cells were isolated from 9-day-old SPF chicken embryos. The cells were grown in Dulbecco’s modified Eagle medium  supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin at 37 °C in a 5% CO reports . AffinitThe relative expression levels of the gp85, p27, TCF4, LEF1, Axin, APC, β-catenin, cyclin D1, c-myc, and 18S were determined by real-time PCR  as reported previously [−ΔΔCT method. The results are presented as fold-change of relative expression compared with the control group.The total RNA from cells was prepared by using the AxyPrep™ Multisource Total RNA Miniprep kit , and 1 μg RNA was used in reverse-transcription reaction using PrimeScript RT Master Mix  following the manufacturer’s instructions. Here, 1 μL diluted cDNA, 400 nM primers, and 10 μL SYBR Green Master Mix were used for the real-time PCR in a final volume of 20 μL. The amplification conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Dissociation curves were generated to analyze the individual PCR products. The gene expression levels were normalized to the level of chicken 18S mRNA. The analyses of the relative gene expression data were performed by using the 2To evaluate β-catenin/TCF transcriptional activity, CEF cells were transfected with the luciferase reporter plasmids pTOP FLASH, which contains wild-type TCF binding sites  and pFOP FLASH, which contains mutant TCF binding sites , as described previously ,20,28. C4/well CEF cells were seeded in 96-well plates, and 100 μL of iCRT14 at 0 and 10 μM in DMEM maintenance medium were added to each well. After incubating for 24, 48, 72, and 96 h in a 37 °C incubator, CCK-8 solution (10 μL) was added to each well. Incubation was continued for 1 h, and the absorbance at 450 nm was measured.The proliferation and cytotoxicity of iCRT14 on CEF and LMH cells were determined using a cell counting kit, CCK-8 kit . In brief, 1.0 × 10As previously reported , the cel5cells/well). Cells were transfected with RNA oligos siRNA 986 or a control siRNA at 140 pmol using Lipofectamine RNAiMax. Then, 24 h after transfection, CEF were infected with ALV-J at MOI of 1.0 and cultured for 72 h. Cell lysates were then collected for Western blot assay, and RNA were extracted for qRT-PCR. The virus titer in the culture supernatant was determined by TCID50 assay.Previously, we demonstrated that the small interfering RNA (siRNA) 986  designed by Genepharma company  showed interference capacity above 50% (unpublished data). In the present study, the CEF cells were seeded in 12-well plates  of triplicate determinations. The significance of the variability between the trials was analyzed using the GraphPad prism (version 5.0) software. Differences in data were evaluated by the Student’s GSK-3 inhibitor X, an acetoxime analog of 6-bromoindirubin-3′-oxime (BIO), has been utilized to activate Wnt/β-catenin signaling by stabilizing β-catenin from the degradation complex in mammalian studies ,29. HoweBy using the Wnt/β-catenin signaling TOP/FOP flash reporter assay, we first validated that GSK-3 inhibitor X could activate the Wnt/β-catenin signaling in CEF cells. In addition, the activation could be restored by iCRT14, one of the specific small molecular inhibitors of β-catenin responsive transcription A,B. Afte50 assay (CEF cells were pre-treated with GSK-3 inhibitor X (5 μM) for 3 h and then infected with ALV-J JS09GY3 at MOI of 1.0. After 72 h of inoculation, the RNA and cell lysates were collected for real-time PCR and Western blotting assay, respectively. We found that the transcription of the gp85 and p27 genes were significantly increased in a dose-dependent manner, compared with the control DMSO group A,B. The 50 assay D. Moreov50 assay E,F. ThesAccording to the results shown in 50 assay. Virus titers in 10μM iCRT14-treated cells were significantly reduced at 72 h p.i., compared to the control group (CEF cells were then treated with gradient concentration (5 and 10 μM) for 4 h before being infected with ALV-J JS09GY3 at MOI of 1.0. After a 72 h incubation, the RNA and cell lysates were collected for real-time PCR and Western blotting assay, respectively. The results showed that the transcription of the gp85 gene were significantly reduced in a dose-dependent manner compared with the DMSO control group A. A simiol group D.In order to further confirm the effect of iCRT14, we also detected the ability of iCRT14 to inhibit virus replication in LMH cells by immunofluorescence and Western blotting assay. To rule out the possibility that the inhibition was due to cytotoxicity of iCRT14 on LMH cells, a CCK-8 cell viability assay was performed. The cell viability was detected by OD450 after treatment with iCRT14 at concentrations of 10 μM A. As expTo further confirm the effects of the Wnt/β-catenin signaling pathway on ALV-J infection, we inhibited the pathway by knockdown of β-catenin using siRNA according to a previous report . In our The Wnt/β-catenin is a crucial signaling pathway in the growth of the body, cell differentiation, communication, and proliferation. It is associated and involved in many diseases, including cancer, neurological, autoimmune, and inflammation diseases ,10. SeveIn mammalian cells, Wnt ligands (Wnt3A and Wnt9B) are usually used to activate the Wnt/β-catenin signaling pathway and induce the expression of target genes. However, our unpublished studies showed that Wnt3A and Wnt9B with different concentrations had no significant effect on CEF cells. Based on the previous study about Wnt/β-catenin signaling pathway in chicken primordial germ cells , we trieIn conclusion, this study provides the first evidence that the activation of the Wnt/β-catenin signaling pathway promotes ALV-J gene expression and virus production in CEF cells, and the inhibition of the Wnt/β-catenin signaling pathway limits virus production in both CEF and LMH cells. These findings provide new insights into the virus–host interaction of ALV-J and identified some potential antiviral strategies for ALV-J control.


Colorectal cancer (CRC) is one of the common causes of cancer death worldwide. Obstructive sleep apnea syndrome (OSAS), sharing many risk factors in common with CRC, is prevalent among CRC patients. OSAS may promote the CRC development independently but the mechanism is still unknown. Intermittent hypoxia (IH) is one of the characteristics of OSAS, and hypoxia may influence the genes associated with CRC. Intestinal microbiota plays important role in CRC carcinogenesis, and OSAS patients have been shown to have intestinal microbiota dysbiosis. We hypothesized that IH and intestinal microbiota dysbiosis may be involved for CRC in patients with OSAS.We established precancerous cell models of CRC with Immorto-Min colonic epithelial (IMCE) cells. First, the cells were exposed to IH in a special chamber for 4 h, 8 h, and 12 h. Feces from 6 patients with OSAS and 6 healthy controls were collected and made into sterile fecal fluid for incubation with IMCE cells for 12 h. The cells were then exposed to IH for 4 h, 8 h, and 12 h. After IH exposure, the expressions of genes and inflammation cytokines associated with CRC, such as β-catenin, STAT3, HIF-1α, IL-6, TNF-α, c-myc, and cyclinD1, were tested.P = 0.015 for mRNA level, P = 0.027 for protein level; STAT3: P = 0.023 for mRNA level, P = 0.023 for protein level), and promoted p-STAT3 shifting to the nucleus (P = 0.023). The mRNA of β-catenin (P = 0.022) and cyclinD1 (P = 0.023) was elevated, but there was no change for the β-catenin protein in the nucleus. Gut microbiota of OSAS patients promoted the expression of STAT3 , promoted p-STAT3  shifting to the nucleus, and also elevated the expression of IL-6 and TNF-α in mRNA level at 4 h  and 8 h . The protein of β-catenin in the nucleus was not affected by IH and gut microbiota from OSAS.IH activated the expression of HIF-1α and STAT3 both in mRNA and protein level (HIF-1α: Our study demonstrated that IH and gut microbiota of patients with OSAS activated HIF-1α expression and STAT3 pathway in IMCE cells, with no influence on β-catenin pathway, which suggested that IH, STAT3 pathway, chronic inflammation, and intestinal microbiota dysbiosis may be involved in CRC carcinogenesis correlated with OSAS These findings must be interpreted cautiously and further research is necessary to clarify the causative steps in CRC development. Colorectal cancer (CRC) is very common worldwide, which lists third in morbidity and second in mortality overall worldwide , and theObstructive sleep apnea syndrome (OSAS), also very prevalent in modern society, is a kind of sleep breathing disorder, with repeated partial or complete upper airway collapse. OSAS is characterized by intermittent hypoxia (IH), microarousal, and sleep fragmentation during sleep , affectiIntermittent hypoxia (IH) is key characteristic of OSAS, and it could induce multiple organ impairment. Continuous hypoxia condition could promote colon cancer cell proliferation and invasion –27, but The human large intestine contains the most microorganisms in the body, which play an important role in absorption, metabolism, and storage of ingested nutrients, with potentially profound effects on host physiology. Intestinal microbiota could also influence the inflammation level through interaction with immunocyte and producing short-chain fatty acid (SCFA) by fermentation. Microbiota dysbiosis has been proved to be associated with CRC . Some spTherefore, we hypothesized that IH and intestinal microbiota dysbiosis in OSAS patients may have the ability to activate one or more CRC carcinogenesis pathways, and inflammation may be involved in this process. To validate our hypotheses, we exposed Immorto-Min colonic epithelial (IMCE) cells to IH and sterile fecal supernatant from OSAS to establish precancerous cell model, mimicking CRC premalignancy in OSAS patients, and the expressions of genes and inflammation cytokines associated with colorectal cancer, such as β-catenin, STAT3, HIF-1α, IL-6, TNF-α, c-myc, and cyclinD1, were analyzed to evaluate the effect of IH and microbiota of OSAS on CRC development.IMCE cell line was preserved in the laboratory of Gastroenterology and Hepatology department , which was kindly provided by Professor Fang Yan from Vanderbilt University. IMCE cells were cultured in RMPI 1640 medium  supplemented with 10% FBS , 0.05% interferon-γ, 100 U/ml benzyl penicillin, and 100 μg/ml streptomycin under a circumstance of 33 °C with 5% CO2, and passaged every 3–5 days. Before IH exposure, the cells were changed into starvation medium  for 12 h. Then, cells were exposed in the experiment room, under intermittent hypoxic condition for 4, 8, or 12 h, and parts of cells were preserved without IH treatment as the control group. IH exposure was in a plexiglas chamber, which was alternately flushed with a hypoxia gas mixture  or normoxia gas mixture  controlled by a computed engine. This IH exposure system for cell experiments was set up earlier by the Department of Respiratory Medicine in Tianjin Medical University General Hospital , and it Fresh feces were collected from 6 OSAS patients (O group) and 6 control healthy people (C group). In each group, 1 g of each sample was taken to mix together. The mixed feces were placed in sterile phosphate-buffered saline (100 mg/ml), homogenized, and centrifuged at 1000 rpm for 1 min, and the supernatant was collected as previously described , 45. Thehttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Real-time PCR was conducted to quantify the transcription of cytokines such as IL-6 and TNF-α, and the transcription of genes associated with colorectal cancer such as HIF-1α, β-catenin, STAT3, cyclinD1, and c-myc. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), known as a housekeeping gene, was used as inner control to normalize the relative expression of targeted genes at mRNA level, which has been validated in some former researches [−ΔΔCt method was used to calculate relative mRNA expression.Total RNA was extracted by Trizol reagent , and cDNA reverse transcription was carried out using the TIANScript RT kit  according to the manufacturer’s instructions. The oligonucleotide primers were synthesized in GENEWIZ . Relevant oligonucleotide primer sequences were shown in Table searches –55. ABI g, 4 °C, 15 min), and the supernatants were retained. The protein was separated by SDS-polyacrylamide gel electrophoresis, then transferred onto a PVDF membrane. Rabbit monoclonal antibodies , anti-β-catenin, anti-HIF-1α, anti-IL-6, anti-p-STAT3, and anti-STAT3 were used correspondingly as primary antibodies. β-actin was used for total protein internal control and Histone 2A for nucleus protein internal control. Goat anti-rabbit IgG conjugated with horseradish peroxidase antibodies  was used as secondary antibody. A chemiluminescence detection system  was used to detect the membranes. Comparison between the intensity of targeted bands and the intensity of internal control band was achieved via an image processor program (Image J 1.51j8).After the IH treatment was finished, the cells were washed with phosphate-buffered saline (PBS) for two times, and then RIPA buffer was used before lysed. Nucleus and cytoplasmic protein extraction kit  was used to extract the protein from the cells. Proteinase inhibitor cocktail (10 μl/ml)  and phosphatase inhibitor cocktail (10 μl/ml)  were added separately. After that, the lysate was homogenized and centrifuged , and increased during the longer IH exposure, but there were no statistically significant differences in the levels between 4 and 8 h IH groups , though with increasing trend, but there were no statistically significant differences in the levels between 8 and 12 h IH groups  , then increased during the longer IH exposure, but there were no statistically significant differences on the levels of 8 h and 12 h IH groups compared with the control group ; though with increasing trend, the levels of 4 h and 8 h group had no statistically significance compared with the control group       Fig. . In prot58) Fig. . Though 23) Fig. , no elev23) Fig. . In anot51) Fig.  and prot98) Fig.  of STAT343) Fig. . These rP < 0.05, F = 4.528)   and 12 h  IH points, and at 8 h IH point, O group’s HIF-1α also had increasing trend but without statistical significance  and 8 h  IH points for mRNA level in O group       and 8 h  IH point   Fig. . At the 99) Fig. , but aftnce Fig. . IL-6 waoup Fig. , but for37) Fig. . TNF-α w42) Fig. . We coul42) Fig.  and prot42) Fig. . STAT3 w44) Fig.  and prot44) Fig. . For p-S30) Fig. . These rint Fig. , and cyc14) Fig. , which iMCE cell line is phenotypically normal, but it has proven to be susceptible to transformation, so it is a very suitable model system available for studying malignant progression in colon cancer . In our Cancer cells proliferate quickly and consume much oxygen, so malignant tumors usually contain hypoxic regions, which is the characteristic of tumor microenvironment. Tumor cells develop corresponding mechanisms for hypoxia environment adaptation. HIFs are activated in hypoxia conditions, regulating the expression of many genes that code for proteins involved in angiogenesis , glucoseChronic IH is the key pathogenesis of OSAS damage. Different from continuous hypoxia, IH is in cycling reoxygenation, and reactive oxygen species could be generated in this progress, which is more similar with ischemia-reperfusion process, so OSAS could be regarded as an oxidative stress disorder . The shiBeside genetic heredity, CRC could be divided into sporadic CRC and colitis-associated cancer (CAC), according to the molecular mechanism by which cancer was triggered and promoted. For sporadic CRC, classical “normal mucosa-adenoma-carcinoma” sequence has been considered to be the core mechanism, and Wnt/β-catenin pathway plays an important role. Once activated, β-catenin would move into the nucleus, regulating its target gene such as cylinD1, c-myc, and MMP-7 which control the cell proliferation . But forAt present, it has been proved that hypoxia and HIFs could influence both Wnt/β-catenin and IL-6/STAT3 pathways. HIF-1 could activate and maintain the Wnt/β-catenin pathway . In HCT Many researches have shown that chronic IH could mimic the OSAS pathogenic process. But only IH itself may not be perfectly suitable for intestine research, because intestinal epithelium is inconstant contact with intestinal microbiota in vivo. It has been proved that the function and disease of intestine are affected by microbiota and their metabolic products. Some research has indicated that intestinal microbiota dysbiosis was induced in OSAS patients , 86 and The microbiota dysbiosis in OSAS patients may correlate to sleep disorders and metabolic comorbidities. OSAS patients with Prevotella enterotype would exhibit worse sleep disruption . CompareOur study showed that both IH and gut microbiota from OSAS patients could promote p-STAT3 entering to the nucleus, but we observed no change for β-catenin pathway in this process. It has been proved that gut microbiota dysbiosis of OSAS patients could induce inflammation, but we only observed elevation of inflammation cytokines for mRNA level in our model, which suggests that our cell model is far from perfect.Our study had many limitations. We did not test the cell proliferation after IH exposure in this study, just tested the genes related to proliferation. Restricted to experiment conditions, it was not accurate for temperature modulation in IH exposure chamber, so the cells were not in good growth conditions during IH exposure, and this would lead to cell viability decrease or even cell death after longtime IH exposure. The outcome would be affected more when IH duration was longer, so we had to restrict IH duration to 12 h at most. Cell proliferation test, such as MTT test, may be meaningless in this condition. The amount of fecal sample was not sufficient enough, so we did not test the composition of feces microbiota in this study. Though we found STAT3 activated, but the pathway upstream is still not clear. Our IH parameters were performed according to previous teamwork , 43, andIn summary, our data demonstrated that IH could activate the expression of HIF-1α, elevate the level of p-STAT3 in the nucleus, and promote the expression of cylinD1 in IMCE cells. Gut microbiota of OSAS patients could increase the expression of STAT3 and elevate the level of p-STAT3 in the nucleus under IH, and promote the expression of IL-6, and TNF-α in mRNA level under IH less than 8 h. Though elevated in mRNA level, β-catenin protein level in the nucleus was not affected by IH treatment and feces from OSAS patients. This suggested that IH, STAT3 pathway, chronic inflammation, and intestinal microbiota dysbiosis may be involved in CRC carcinogenesis correlated with OSAS. But this conclusion is very limited, still long distance to clarify the CRC development.


The inhibition of Wnt/β-catenin remarkably reversed the influence of shZFAS1 on cardiac function and cardiac fibroblasts viability. Therefore, Knockdown of ZFAS1 could improve the cardiac function of myocardial infarction rats via regulating Wnt/β-catenin signaling pathway. The present study might provide new thoughts for the prevention and treatment of MI damage.Myocardial infarction (MI) is a big health threat in the world, and it is characterized by high morbidity and mortality. However, current treatments are not effective enough, and novel therapeutic strategies need to be explored. ZFAS1 has been proved to be involved in the regulation of MI, but the specific mechanism remains unclear. MI rats were constructed through left anterior descending artery ligation, and hypoxia cell model was also established. The proliferation, invasion, and migration of cells were detected via CCK8, traswell, and wound healing methods. Immunohistochemistry staining, western blotting, and qRT-PCR were used to detect the levels of molecules. Knockdown of ZFAS1 significantly increased the proliferation, migration, and invasion of cardiac fibroblasts. Knockdown of ZFAS1 remarkably improved cardiac function via decreasing infarction ratio and increasing vWF expression, left ventricular ejection fraction, and left ventricular fractional shortening compared with group MI. Knockdown of ZFAS1 also suppressed Wnt/β-catenin pathway  Myocardial infarction (MI) is a kind of myocardial necrosis caused by acute ischemia and hypoxia of coronary artery. If the coronary artery is blocked for more than 40 minutes, the myocardium will be damaged irreversibly , 2. Some2+ ATPase 2A (SERCA2a), which can induce intracellular calcium overload in myocardial cells and further lead to systolic and diastolic dysfunction after myocardial infarction [Long non-coding RNA (LncRNA) is a class of RNA molecules with transcripts longer than 200 bp and without the ability to encode mature proteins. A large number of studies have shown that LncRNA is involved in the occurrence and progression of tumor, diabetes, cardiovascular disease and other diseases , 7. Zincfarction , 12. In farction  and ischfarction . TherefoWnt/β-catenin signaling pathway is involved in embryo and organ development. Wnt family genes mainly encode secretory signaling proteins, which are related to tumorigenesis and adipogenesis. In addition, it is related with the regulation of cell differentiation during embryonic development –17. It wIn this study, MI rats and hypoxia cell models were established. Knockdown of ZFAS1 was constructed and the influence of ZFAS1 on the cell viability and cardiac function were investigated. Meanwhile, the effect of ZFAS1 on the Wnt/β-catenin pathway and if ZFAS1 could regulate MI through Wnt/β-catenin were also studied. This study provides a new insight for the prevention and treatment of MI damage.in vivo and in vitro models were established. Significant increase of ZFAS1 in both MI and hypoxia models was observed  could remarkably suppress the levels of Wnt3a and β-catenin in myocardial tissue, but the down-regulation was significantly suppressed by the inhibitor of Wnt/β-catenin pathway. Silencing PEDF can suppress vascular endothelial injury by inhibiting Wnt/β-catenin signaling pathway, so as to reduce the cardiac function damage after MI , 30. GSKIn summary, we demonstrated that significant improvement of cardiac function in the MI rats was observed through knockdown of ZFAS1. In addition, we proved that ZFAS1 might regulate the process of MI through activating Wnt/β-catenin pathway. These findings were validated through MI rats and hypoxia cell models. This study uncovers the underlying regulation mechanism of ZFAS1 in MI process and provides a new insight for the prevention and treatment of MI.2. Hypoxia cell model was established by incubating cells on the condition of 93% N2, 3% O2, and 4% CO2 for 24 h. Then, the cells were incubated with shZFAS1, shNC, and XAV939 for 12 h. Then, cells were used to measure cell proliferation, migration, and invasion. All experiments were approved by Ethic Committee of The Second Affiliated Hospital of Nanchang University .Cardiac fibroblasts (CFs) were obtained from American Type Culture Collection . Normal CFs were incubated with DMEM containing 10% FBS in the incubator of 37°C and 5% CO13 genome-containing particles) was used to treat rats through a caudal vein.AAV9 vector was used to carry a short RNA fragment to knockdown ZFAS1 (shZFAS1). The shZFAS1 and shNC were constructed by GenePharma Co., Ltd . Virus solution  were used in this study. The animals were kept in the condition of 25–28°C, 45–55% humidity with free access to food and water. The rats were divided randomly into different groups. MI model was established by performing left anterior descending ligation using 6.0 nylon. The rats were firstly anesthetized through intraperitoneal injection with xylazine (11 mg/kg) and ketamine (110 mg/kg). After shaving the chest of rats, a left parasternal incision was performed to open thoracic cavity. Then, heart was exposed, and left anterior descending was ligated for 1 h. Remarkable elevation of S-T segment in electrocardiograph indicated MI. Same operation was conducted for the sham group rats excluding the ligation of left anterior descending artery. 1 week after operation, rats were injected with shZFAS1, shNC, or XAV939. 2 weeks later, rats were sacrificed and heart tissues were collected for histological examination.RNA was isolated through TRIzol reagent . 30 ng RNA was reverse-transcribed into cDNA with SuperScript™ II Reverse Transcriptase . RT-PCR was performed using SYBR Premix Ex Taq™ II kit . The primers were listed as follows: (1) β-catenin: forward: 5′-TTCGCCTTCACTATGGA CTACC-3′ and reverse: 5′-GCACGAACAAGCAAC TGAACTA-3′; (2) GSK-3β: forward: 5′- CGAUUACACGUCUAGUAUA -3′ and reverse: 5′- UAUACUAGACGUGUAAUCG -3′; (3) GAPDH: forward: 5′-ACAACAGCCTCAAGATCATCAG-3′ and reverse: 5′-GGTCCACCACTGACACGTTG-3′. The mRNA level of the experimental group was determined by comparing the value of ΔΔCt.Tissues were lysed firstly, and protein content was detected using bicinchoninic acid kit  assay. Same amount of protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membrane . The membrane was blocked with TBST solution (5% skim milk) for 2 h, and cultured with primary antibodies (1:800) at 4°C overnight. After washing twice using PBS, a secondary antibody (1:2000) was used to incubate proteins for 1 h. ImageJ software was applied to calculate the protein bands grey. The antibodies details were listed as follows: anti-beta Catenin antibody , anti-GSK3 beta antibody , and goat anti-Rabbit IgG .6 each well) were seeded into the upper chamber, and 2 mL DMEM containing 15% FBS was added to the down chamber. The cells were cultured for 24 h, then 4% polyformaldehyde was used to fix cells for 20 min. After washing twice, Giemsa  was used to stain lower chamber. The invasive cells in the five fields were captured and analysed.Cells . Rats were firstly anesthetized using xylazine (11 mg/kg) and ketamine (110 mg/kg). Then hemodynamic items were recorded through MP100-CE .The tissues were fixed with 4% paraformaldehyde for 24 h. Then, tissues were embedded with paraffin and cut into 8-μm thick slides. The tissues were stained through hematoxylin-eosin (HE) and Masson trichrome methods, respectively. Five fields were selected for analysis. An inverted optical microscope was used for analyzing.2O2 (5 min). After blocking with 10% goat serum, the slides were cultured with primary antibody at 4°C overnight. After washing , the sections were cultured with secondary antibody for 1 h. Then DAB regent was applied to incubate sections, and an inverted microscope was used for analyzing.After de-paraffin, microwave heating was applied to repair antigen. Then, tissues were washed , and cultured with 5% H5) were plated into 96-well plate and cultivated for 24 h. After different treatments, cells were incubated with MTT reagent (20 μL) for 4 h, OD at 490 nm was detected.MTT assay  was applied to detect cell proliferation. Cells , and analyzed through SPSS 20.0. Student's