Hibiscus cannabinus L.) and Bamboo fibre, together with multi-walled carbon nanotubes (MWCNTs) as reinforcing fillers in the epoxy matrix. Mechanical testing revealed that hybridization of natural fibres was capable of yielding composites with enhanced tensile properties. Additionally, impact testing showed a maximum improvement of ≈80.6% with the inclusion of MWCNTs as nanofiller in the composites with very high energy absorption characteristics, which were attributed to the high specific energy absorption of carbon nanotubes. The viscoelastic behaviour of hybridised composites reinforced with MWCNTs also showed promising results with a significant improvement in the glass transition temperature (Tg) and 41% improvement in storage modulus. It is worth noting that treatment of the fibres in NaOH solution prior to composite fabrication was effective in improving the interfacial bonding with the epoxy matrix, which, in turn, resulted in improved mechanical properties.Recently, there has been an inclination towards natural fibre reinforced polymer composites owing to their merits such as environmental friendliness, light weight and excellent strength. In the present study, six laminates were fabricated consisting of natural fibres such as Kenaf fibre ( Research on natural fibre reinforced composites has been growing at a relentless pace as it holds the promise of delivering composites with low density (weight) yet high specific properties ,2,3,4,5.Cyrtostachys renda (CR)-Kenaf fibre-based, hybrid polymer composites were fabricated [Today, as natural fibre reinforced composites are gaining more interest in various applications, research has been diverged towards developing hybrid composites to achieve properties such as ductility, stiffness and strength, which are non-attainable in a single fibre reinforced composite . Besidesbricated . The tenbricated . In all Additionally, the last few decades have witnessed significant research expansion on polymer nanocomposites using various carbonaceous nanofillers such as graphene and carbon nanotubes (CNTs). For instance, CNT as nanofillers in polymer composites have been reported to exhibit high structural and functional properties owing to their high aspect ratio, electrical properties and mechanical strength ,16. In a(Hibiscus cannabinus L.), a type of bast fibre with high aspect ratio compared to other fibres with superior toughness, strength and stiffness [In the present study, Kenaf fibre tiffness  along witiffness ,16 were tiffness ,18,19. TBambusa vulgaris) mats and Kenaf (Hibiscus cannabinus) mats in 1.2 g/cm2 were purchased from a local supplier in Chennai, India. To prevent defects and improve the interfacial adhesion between the fibre and Epoxy, alkali treatment was performed using 5% of NaOH. The Bamboo and Kenaf fibres were then cleaned using distilled water and dried at room temperature. MWCNTs (the reinforcing fillers); NC7000 used in the present study in the form of black powder and spaghetti-like structure at nanoscale with 98% purity, with an average cross-sectional diameter and length of 10 nm and 6–9 µm, respectively, was synthesised using the catalytic chemical vapor deposition method (CCVD) by NANOCYL, Belgium. Laminates having dimensions of 270 mm × 270 mm × 3 mm were prepared at a pressure and temperature of 2500 psi and 230 °C, respectively, using a compression moulding machine (100 tonne capacity). Cooling was undertaken at room temperature. Six laminates of natural fibre reinforced composites were prepared as shown in The current research used Bamboo and Kenaf fibres and multiwall carbon nano-tubes (MWCNT) to reinforce Epoxy resin. In this regard all different combination of fillers were attempted to investigate the effect of single filler reinforcement and hybridization as well. Epoxy HY951 and Aradur 22962 hardener which were used to bind the fibres (in random) were supplied by Herenba Instruments and Engineers, Chennai, India. Untreated Bamboo  standards. Tensile testing was carried out using a universal testing machine  equipped with a 100 kN capacity load cell. Both the ends of the samples were tabbed to avoid slipping. The respective tensile properties were measured for each laminate. Energy absorbed by each laminate was tested using an impact testing machine  according to ASTM D256 standards. To evaluate the transverse loading ability of the prepared laminates, a 3-point bending test was carried out with samples (100 mm × 20 mm × 5 mm) according to ASTM D-790. Dynamic mechanical analysis (DMA) was performed using DMA Q800  with the 3-point bending clamp setup. The analysis was carried out on a temperature sweep mode from 30 °C to 180 °C at a frequency of 0.1 Hz, preload force of 0.01 N and amplitude of 15 µm. Unless otherwise stated, 3 samples were fabricated and tested for each laminate condition to ensure reproducibility of test results.Thermo gravimetric analysis (TGA) was carried out using Netzsch STA 449F3 instrument  to evaluate the expansion and contraction of the laminates due to fibre swelling from moisture absorption. It was undertaken in an alumina crucible that was heated in flowing air (260.3 mL/min) and nitrogen (250.0 mL/min) from 35 °C to 1000 °C at a heating rate of 10 °C/min. The vibrational spectra of the prepared composites were recorded using Fourier transform infrared (FTIR) spectrometer  while the viscous behaviour of the laminates having dimensions of 10 mm × 10 mm × 4 mm was measured using the cantilever method from 25 °C to 168 °C at a heating rate of 100 °C/min and maximum frequency of 5Hz. Scanning electron microscopy (SEM) was performed using JSM-7600F  with an accelerating voltage of 2–5 kV on the surfaces of the fractured surfaces. The samples were first coated with ~4 nm of Pt using Hitachi S-3000N machine and examined to understand the microstructure of the broken samples. Raman spectroscopy analysis was performed using stellar-Pro ML150 laser coupled to an optical microscope (Leica DM 2500 M) with Renishaw He-Ne laser with 633 nm wavelength as excitation source.The combined TGA and DTG data from 35 °C to 800 °C of the six prepared laminates are shown in −1 region represents the O–H stretching vibrations of the cellulose fibres such as Bamboo and Kenaf [−1), C=O stretching (1641 cm−1), aromatic ring , C-H deformations, C-O stretch (1242 cm−1), C-O stretching of lignin, ester groups (1180 cm−1), C-O deformations (1018 cm−1) and p-hydroxyphenyl C-H out of plane . Similar findings have been reported in other studies [−1 [, which ascertains the interaction (adhesion) of MWCNT within the composites. All the spectra of these composites have confirmed the compositions and interaction between the Bamboo, Kenaf and natural cellulose functional groups with the Epoxy resin. Besides, the polymerization of Epoxy resin and product oxidation were confirmed by the absence of Epoxy resin’s significant peak at 915 cm−1. A detailed FT-IR spectrum of all laminates labelled with their respective vibrational bands are shown in the The vibrational frequencies from FT-IR analysis and the resultant peak values are shown in nd Kenaf . The com studies ,24. MWCNdies [−1 , which a−1 and 1128 cm−1 were assigned to the resin backbone vibrations, whereas Raman bands corresponding to Epoxy ring deformations and Epoxy ring breathing were located at 690 cm−1 and 1251 cm−1 respectively [−1 can be assigned to the amide I vibration, predominant of C=O stretching of the amide group [−1 can be associated with the highly crystalline cellulose structure  originating from Bamboo and Kenaf fibres. Similar band have been reported for Avicel (SA21) Valonia and tunicate (SA18) celluloses [−1 and 1530 cm−1, which correspond to the D and G-bands, respectively, and are consistent with typical characteristics of graphitic MWCNTs [2, which shows that the nanotubes are highly graphitised. The intensity of the D-band to G-band (ID/IG) semi-quantitatively indicate the structural integrity of MWCNTs in the Epoxy matrix. In the present study, the ID/IG ratio was 1.12, which ascertains that the MWCNTs were not imposed to significant amount of defects [The structural changes on the molecular level of MWCNT based Epoxy composites were investigated using Raman spectroscopy. From ectively ,27. The lluloses . Raman s defects .2 whereas lowest tensile strength was observed in laminate 1 (Bamboo + Epoxy) with a value of 45.85 N/mm2. Similar high-tensile strength value of Kenaf + Bamboo fibre + MWCNT reinforced Epoxy composites were also reported in other studies [The stress-strain curves of the laminates are shown in  studies ,13,32. I studies . Besides2, even without the addition of MWCNT fillers. The addition of MWCNTs elevated the strength of the composite by 3.7%, which can be attributed to the efficiency of load transfer and the interfacial bonding between MWCNT, other fillers and the Epoxy matrix. The high surface area per unit volume of MWCNT led to a good interfacial bond. which is essential to ensure the effective stress transfer from the Epoxy matrix to the fibres, through which the fibre’s strength can be utilised to its full potential in the composite ,35. The 2 and 14.4 J/mm2 respectively. The slightly higher impact strength of Kenaf reinforced composite can be attributed to the woven Kenaf structure compared to the non-woven Bamboo; where woven structure fibres render better impact strength than the random fibres orientation [Izod’s impact test was conducted on the composite samples to ascertain their capability of withstanding sudden load. From entation . Upon hyIn a study by Shirvanimoghaddam et al., it was reported that for polymer blends, factors such as interparticle distance or matrix ligament thickness determines the brittle to tough transition of the polymer blend . As such−3 [−3 .Flexural strength of a material is referred to as the maximum bending stress required to break a beam under a three-point bending load. The flexural behaviour of composites fabricated in the present study is shown in + Bamboo + Epoxy + MWCNT). Phenomena such as fibre breakage, pull-outs, voids and matrix cracking were all clearly observed from these figures. From Glass transition temperature (Tg) of the laminates were investigated using DMA which was determined by the maximum peak of tan δ. It is clearly evident that the Tg of laminates increased from 83.3 °C to a maximum value of 110.2 °C when the Epoxy matrix was reinforced with Kenaf, bamboo and MWCNTs. Besides, it is also observed that the Tg of laminates 4, 5 and 6  were higher than laminate 1, 2 and 3 . This is a clear indication that the incorporation of MWCNTs have played a significant role in increasing the Tg of the composites. The increase in Tg after incorporation of MWCNTs is attributed to the reinforcement effect, which tightens the whole network and hinders mobility of polymer chains, an indication of good adhesion between the MWCNTs, bamboo fibre, Kenaf fibre and the Epoxy matrix. Similar findings have been reported in other studies with the incorporation of MWCNTs as reinforcement filler ,46.In conclusion, hybridization of natural fibre with reinforcing effect from MWCNTs in Epoxy composites showed improved mechanical properties. For instance, maximum tensile strength of 67.3 MPa was achieved in Kenaf + Bamboo + Epoxy + MWCNT composite. Hybridization of high elongation fibres with low elongation fibres in the Epoxy matrix improved the strain level required for propagation of fibre breakage where higher elongation fibre was capable of arresting the crack at micromechanical level. Besides, Kenaf + Bamboo + Epoxy + MWCNT hybrid composites showed the best resilience to sudden shock with excellent energy absorption characteristics in comparison to other composites attributed to the excellent load transfer efficiency of MWCNT. Furthermore, incorporation of MWCNTs played an essential role in increasing Tg of the composites by providing the tightening effect to the whole network, which, in turn, hinders mobility of the polymer chains. The insights derived from our study indicate that the hybridization of natural fibres in Epoxy matrix, together with the addition of MWCNTs as nanofiller reinforcement has the potential to be a leading material for applications requiring high strength and excellent impact energy absorption.


Canonical Wnt/β-catenin signaling is a complex cell-communication mechanism that has a central role in the progression of various cancers. The cellular factors that participate in the regulation of this signaling are still not fully elucidated. Lysine acetylation is a significant protein modification which facilitates reversible regulation of the target protein function dependent on the activity of lysine acetyltransferases (KATs) and the catalytic function of lysine deacetylases (KDACs). Protein lysine acetylation has been classified into histone acetylation and non-histone protein acetylation. Histone acetylation is a kind of epigenetic modification, and it can modulate the transcription of important biological molecules in Wnt/β-catenin signaling. Additionally, as a type of post-translational modification, non-histone acetylation directly alters the function of the core molecules in Wnt/β-catenin signaling. Conversely, this signaling can regulate the expression and function of target molecules based on histone or non-histone protein acetylation. To date, various inhibitors targeting KATs and KDACs have been discovered, and some of these inhibitors exert their anti-tumor activity via blocking Wnt/β-catenin signaling. Here, we discuss the available evidence in understanding the complicated interaction of protein lysine acetylation with Wnt/β-catenin signaling, and lysine acetylation as a new target for cancer therapy via controlling this signaling. Canonical Wnt/β-catenin signaling is one of the well-known conserved cell-communication mechanisms that involve the growth, metastasis, stemness maintenance, and therapeutic resistance in different kinds of cancer , 2. EspeAs an evolutionarily conserved protein modification, lysine acetylation can transfer the acetyl group from acetyl-coenzyme A to target substrates to alter their biological functions . Until nIncreasing evidence indicates that the protein lysine acetylation, including the acetylation of histones as well as non-histone proteins, is vital for Wnt/β-catenin signaling activation. Conversely, this signaling can regulate the protein lysine acetylation. Here, we discuss the latest advances related to protein lysine acetylation to regulate Wnt/β-catenin signaling, the effect of this signaling on controlling protein lysine acetylation, as well as the potential of targeting lysine acetylation to inhibit this signaling to facilitate cancer treatment.As mentioned above, the effect of Wnt/β-catenin signaling in different biological processes is mainly dependent on Wnt target genes . HoweverIn Wnt/β-catenin signaling, as a restriction factor, GSK3β also could regulate the acetylation levels of different molecules. Eom et al. reported that GSK3β can bind to p53, and the interaction is capable of initiating K373 and K382 acetylation in p53 . HoweverSo far, in Wnt/β-catenin signaling, the acetylation of four molecules, including LRP6, TCF4, GSK3β, and β-catenin has been unveiled Fig. . For exaTo date, β-catenin acetylation has been widely explored by different groups. It has been found that β-catenin acetylation is relevant to CBP, p300, and PCAF. Especially, the acetylation of K345 in β-catenin is associated with p300 . The K49Moreover, dependent on HATs as mentioned above, multiple molecules are involved in modulating β-catenin acetylation. For instance, Li et al. found that blocking proliferation 1 (BOP1) can initiate β-catenin acetylation that is dependent on CBP to strengthen the drug resistance of breast cancer . ForkheaAlthough several molecules mentioned above contribute to β-catenin acetylation, current reports show that other cellular factors can inhibit the acetylation of β-catenin by suppressing KATs. For example, the transcription factor Kruppel-like factor 4 (KLF4) is critical for intestinal differentiation. Moreover, the differentiation mediated by KLF4 is observed to rely on its interaction with β-catenin to inhibit acetylation of the protein mediated by p300/CBP . The nucApart from KATs, many molecules are capable of restricting β-catenin acetylation by KDACs, including SIRT1, SIRT2, HDAC1, HDAC4, HDAC6, and HDAC7. For example, in bladder cancer cells, Capsaicin can attenuate cell migration via SIRT1-dependent inhibition of β-catenin acetylation . AdditioRecently, Chen et al. reported that B-cell lymphoma 3 (Bcl-3) can maintain K49 acetylation in β-catenin. Mechanistically, Bcl-3-dependent suppression of interaction between HDAC1 and β-catenin is associated with increased β-catenin acetylation in colorectal cancer . Also, HAs mentioned, after Wnt/β-catenin signaling sensitization, β-catenin can interact with and activate TCF/LEF, which is also controlled by various transcriptional coregulators, including the coactivators and corepressors, at the target gene promoter in the cell nucleus , 47. WitUpon activation by Wnt molecules, β-catenin is capable of interacting with TCF/LEF and disrupting the interaction of TCF/LEF with HDACs to form an active transcriptional complex in the cell nucleus Fig. . MeanwhiHowever, in the nucleus, BTB domain and CNC homolog 1 (Bach1) is found to directly bind to TCF4, and the interaction not only reduces the binding of β-catenin to TCF4 but also decreases the interaction of p300/CBP with β-catenin to inhibit β-catenin acetylation. Furthermore, Bach1 can occupy the TCF4-binding site and recruit HDAC1 to the target genes promoter . BarH-liTo date, increasingly studies have indicated that the activation of histone acetylation at Wnt, β-catenin, and FZD promoters can control their transcription Table . In detaIn contrast, histone acetylation at several endogenous inhibitory proteins, including WIF-1, SFRP, and DKK1, can regulate the expression of these molecules to inhibit Wnt/β-catenin signaling. For example, WIF-1 expression can be silenced by suppression of histone acetylation. Following treatment with trichostatin A (TSA), the expression of WIF1 is restored. Furthermore, HDAC3 may contribute to the restriction of histone acetylation at the promoter of WIF-1 to block its expression . In HCC In breast cancer, it was found that prostate tumor overexpressed-1 (PTOV1) can suppress transcription of DKK1 by recruiting HDAC1 and HDAC2 and decreasing histone H3/H4 acetylation levels at the DKK1 promoter . Li et aGiven that KATs and KDACs can modulate protein acetylation, compounds with the function of blocking the activity of KATs or KDACs have important therapeutic potential for diseases involving dysfunction of protein acetylation. To date, numerous KAT inhibitors and KDAC inhibitors have been discovered, and some of these inhibitors have been approved for tumor treatment or undergone clinical trials to explore their exact clinical effect on a variety of cancers , 71. OurCurcuma longa L, with the function of targeting P300, could decrease the growth of HCC cells through regulating the Wnt/β-catenin pathway. Moreover, the clinical applications of curcumin in suppressing multiple cancers, including breast cancer, colorectal cancer, and pancreatic cancer have undergone Phase I/II clinical trials [Garcinia indicia [Consistent with the above speculation, current data indicate that some KAT inhibitors can suppress this signaling Table . Especial trials . Garcino indicia , 75, als indicia –86.TableFurthermore, several histone deacetylase inhibitors, including TSA , 87–89, The canonical Wnt/β-catenin pathway is a conserved signaling mechanism that modulates a variety of physiological and pathological processes. Especially, canonical Wnt/β-catenin signaling is often hyperactivated in cancers and has a significant role in the occurrence and progression of the disease , 97, 98.As mentioned, protein lysine acetylation involves histone acetylation and non-histone acetylation. Histone acetylation is a vital epigenetic process that critically facilitates the control of gene expression. Additionally, as a very important type of PTM, non-histone acetylation can influence the expression and activity of target proteins. The reviewed studies presented here reveal that Wnt/β-catenin signaling has a significant role in the modulation of two types of protein lysine acetylation. Conversely, protein lysine acetylation not only modulates the expression of important molecules in this signaling by histone acetylation but also directly regulates the function of these signaling-related core proteins to control its activation. To our knowledge, although the available data have demonstrated the cross-regulation between this signaling and protein lysine acetylation as mentioned above, our information on the detailed interaction between the constituents of Wnt/β-catenin signaling with protein lysine acetylation, including histone acetylation as well as non-histone protein acetylation, remains limited. Therefore, additional research focusing on the interplay of Wnt/β-catenin signaling with protein lysine acetylation is required to deeply understand their coordinated roles and related mechanisms.Generally, protein lysine acetylation relies on the balance of catalytic activity in KATs and KDACs. So far, a variety of KAT inhibitors and KDAC inhibitors have been identified , 71, and


In vivo, fluorescent xenogeneic endometriosis model was established. In vitro, invasion and metastasis were analyzed after treating FLT. Epithelial-mesenchymal transformation and Wnt/β-catenin pathway were inspected in vitro and in vivo. Activator or inhibitor of Wnt/β-catenin signaling was performed to inspect mechanism of epithelial-mesenchymal transformation. In vivo, FLT not only decreased fluorescent intensity and volume of ectopic lesion, but also ameliorated pathological morphology. E2 and PROG levels in serum were reduced by FLT. In endometrial cells, FLT significantly inhibited the invasion and metastasis. Meantime, epithelial-mesenchymal transformation was reversed, accompanied by suppression of Wnt/β-catenin pathway. In-depth study, activation of Wnt/β-catenin pathway lead to promotion of epithelial-mesenchymal transformation, which was reversed by FLT. FLT prevented fluorescent allograft endometriosis and endometrium cells, which was related to suppress epithelial-mesenchymal transformation through inactivating Wnt/β-catenin pathway. The findings disclose molecular mechanism of epithelial-mesenchymal transformation in endometriosis by FLT, and contribute to further application.Previously the potential therapeutic action of ferulic acid, ligustrazine and tetrahydropalmatine (FLT) are discovered with unclear mechanism in rat autograft endometriosis. However, the effect of FLT on endometrial cells and allograft endometriosis is still unclear. This study is designed to elucidate the influence of FLT on epithelial-mesenchymal transformation in allograft endometriosis and endometrium cells.  Epithelial-mesenchymal transformation is the cell process from epithelial to mesenchymal phenotype, then acquisition of invasion and metastasis. In epithelial-mesenchymal transformation, E-cadherin attenuates as one of the epithelial markers. As mesenchymal markers, N-cadherin, Vimentin, Snail, ZEB1, Twist, and Slug expand for mesenchymal phenotype Foshou san has the effects of invigorating blood and fusing stasis Foshou san. Both of them join with tetrahydropalmatine to form FLT, a new Chinese herbal monomer recipe. FLT has displayed the conceivable reaction on autograft EMS In Chinese medicine, EMS belongs to blood stasis syndromes, especially based on the blood stasis and obstruction of uterus in vivo and in vitro. Using endometrial cells, scratch test and transwell assays were performed after treatment with FLT. Regulations of epithelial-mesenchymal transformation and Wnt/β-catenin signaling were detected by FLT in endometrium cells and allogenic EMS.In the study, therapeutic effect of FLT was observed mT/mG mice were kindly provided by Pro. Lin Chen of Army Medical University from Jackson Lab. 18-22 g C3H mice were supplied for cross breeding . 18-22 g female nude mice were used for allograft EMS . The operation of mice was approved by Care and Use of Laboratory Animals of Southwest University (Authorization 201702). Anesthesia and other necessary methods were provided to reduce suffering.ROSAFLT were composed of 99.8% ferulic acid, 99.3% ligustrazine, and 98.1% tetrahydropalmatine , with the ratio of 1:0.5:0.3. FLT were dissolved at 0.5% CMC-Na or DMSO for mice administration or endometrial cell treatment separately. The positive control selected gestrinone . LiCl  or XAV-939  was provided as activator or inhibitor of Wnt/β-catenin pathway.mT/mG mice. Then, 4 mm2 fluorescent uteruses were transferred to subcutaneous abdomens in nude mice. After operation, nude mice were intramuscularly administrated with 2 mg·Kg-1 estradiol in 5 days' interval. 28 days later, ectopic lesions were captured by Fusion-FX7 imaging system . Fluorescent intensity more than 9.58E+6 were regarded as successful EMS model. According to fluorescent intensity, EMS mice were randomly into 5 groups. Firstly, EMS group was treated with CMC-Na. Secondly, there were 3 FLT groups using 90, 180, and 360 mg·Kg-1 FLT. Finally, another group was treated with 2 mg·Kg-1 gestrinone. Then, no operation mice were performed for control with same treatment as EMS group. In the end of 28 days' treatment, Evolution-Capt software  was processed to investigate and analyze fluorescence intensity of ectopic lesions. Meanwhile, cubage of xenogeneic EMS was estimated with vernier caliper.Allograft operation was slightly modified from the previous studies Eutopic and allograft endometrium were gathered from control and other 5 groups separately. Then tissues were marked with hematoxylin and eosin after fixation with paraformaldehyde. Endometrial morphology was observed in the microscope .According to ELISA kit instruction, prepared serums were added into the equilibrated plates with dilution reagents. Then the plates were annexed with HRP-conjugate reagents, and incubated for 30 min. After incubating with chromogenic reagent, the plates were processed to stop reaction. Then absorbency was detected at 450 nm in spectrophotometer reader .2.Pro. Xiao-hong Chang of Peking University provided hEM15A cells, the human endometrial stromal cells from EMS patients 4 endometrial cells were cultured in each well. After nearly 80% cell convergence, 1 ml pipette tip were utilized for wounded traces in plate. Treated with different doses of FLT, microplates were detected under 50 × magnification in different time points. Image software  was utilized for calculation of scratch size. Migration rate = (average scratching size in 0 h - average scratching size in 24 h)/average scratching size in 0 h × 100%.In 24-well microplate, 6-9×104 endometrial cells were cultured in each well with serum-free FLT medium in transwell inserts . The lower chambers of 24-well plate were distributed with 5% serum medium. Then plate with transwell inserts were incubated at 37 °C for 24 h. The bottom of transwell inserts were dyed with gentian violet for observation of infiltrating cells excluding surface cells. Under the microscope, 5 vision were randomly observed for infiltrating cell count.3-5×10 in vivo and in vitro. Then PrimeScript™ RT Reagent Kit was purchased from Takara (China) for cDNA synthesis. SYBR™ Mix of Thermo Fisher (USA) was applied for gene expressing with 2-∆∆CT calculation. Primer sequences of mRNA were produced by Shenggong Biotechnology   was utilized RNA isolation, China) . β-actinProteins of allografts and cells were processed to western blotting following previous method P values, less than 0.05 threshold, was regarded as statistical difference.One-way ANOVA approach was performed for data analysis using SPSS 21.0 software. After allotransplantation for 28 days, 25 of 30 nude mice were testfied fluorescent allograft EMS with 83.33% success rate. Before treatmeant, distinct difference of fluorescent intensity were not found in EMS, FLT, and gestrinone groups. After treating for 28 days, the fluorescent intensity showed no diversity compared to pretreatment in EMS group. Using FLT, remarkable decrease of fluorescent intensity were detected in recipient nude mice Fig. A, B. The-1 FLT, pathological morphology of ectopic lesion were ameliorated, accompany with less and smaller ectopic glands, fewer microvascular and inflammatory infiltration . Different concentrations of FLT expressed the different effect, which were lower in 90 mg·Kg-1 group, and higher in 180 or 360 mg·Kg-1 groups   Fig. A-D. Cellrol Fig. E-H. All -1 FLT. The mRNA levels of N-cadherin and Vimentin obviously attenuated in FLT groups compared to control group (Fig. Epithelial-mesenchymal transformation is correlated to invasion and metastasis oup Fig. A. Meantioup Fig. B. Moreovoup Fig. C. At theoup Fig. D. The da-1 FLT. Treated with 480 μg.mL-1 FLT, the mRNA level of CyclinD1 were significantly down-regulated (Fig. -1 FLT increased the expression of GSK3β gene in HEC1-B cells, while 1250 μg.mL-1 FLT reduced β-catenin mRNA level. The gene expression of CyclinD1 became less in 625 and 1250 μg.mL-1 FLT groups than control group (Fig. After using FLT for 24 h, there was no obvious effect of FLT on GSK3β gene expression in hEM15A cells. mRNA level of β-catenin gene was remarkably reduced especially using 480 and 960 μg.mLDuring canonical Wnt signaling, β-catenin is subsequently degraded after phosphorylated by GSK3β XAV-939, a tankyrase inhibitor, can increase phospho-β-catenin through inducing degradation via Wnt/β-catenin pathway.In this experiment, FLT markedly inhibited fluorescent ectopic issue growth, E2 and PROG levels, ameliorated pathological change in allograft EMS model. Meanwhile, FLT prevented invasiveness and migration in endometrial cells, which was correlated to repress epithelial-mesenchymal transformation in vitro. In depth study, after treated with FLT, epithelial-mesenchymal transformation was prevented through ascendant E-cadherin, descendant interstitial biomarkers, such as N-cadherin, Vimentin, Snail, Slug, Twist, and ZEB1 in vitro. These data indicated the underlying mechanism of FLT on invasiveness and migration may be related to inhibit epithelial-mesenchymal transformation.Nowadays, EMS has the high morbidity in reproductive age women with equivocal pathogenesis. Recognized phenomenon recovers that implantation of active endometrium cells mainly depends on invasion and metastasis in menstrual reflux hypothesis The canonical Wnt signaling is prevalent in development, differentiation, and proliferation Ferulic acid, ligustrazine and tetrahydropalmatine are the components of FLT, the novel Chinese herbal monomer recipe. In previous research, ferulic acid represses metastasis through withdraw epithelial-mesenchymal transformation in breast cancer FLT prevented invasiveness and migration in endometrium cells, and ectopic growth of allograft EMS. FLT suppressed epithelial-mesenchymal transformation through inhibiting Wnt/β-catenin signaling. These results reveal the pharmacologic mechanism for FLT further research.Supplementary tables.Click here for additional data file.


Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non‐histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance‐7 (MCM7) as the direct PRMT5‐binding partner. A co‐immunoprecipitation (co‐IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1‐248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non‐cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC. We explored the oncogenic roles of PRMT5, and our results showed that PRMT5 induced CRC cell proliferation, migration and invasion in vitro and in vivo. We revealed the physical interaction between PRMT5 and the minichromosome maintenance‐7 (MCM7) protein with a co‐immunoprecipitation (co‐IP) assay, and the direct binding domain was found to be located between residues 1‐248 in MCM7. The proliferative, migratory and invasive properties of CRC cells were greatly impaired after MCM7 silencing in vitro. Furthermore, we evaluated the PRMT5 and MCM7 protein levels in 99 CRC and 77 adjacent non‐cancerous tissues by immunohistochemical (IHC) analysis. These results indicated that PRMT5 and MCM7 were upregulated in CRC compared with normal control tissues. Our results also showed a positive correlation between PRMT5 and MCM7 expression. In addition, we found that PRMT5 could be used to predict for CRC patient outcomes and that patients with high PRMT5 expression among total CRC patients and in the subgroup with a tumour size of ≤5 cm had unfavourable outcomes. In addition, bioinformatic analysis of CRC data from TCGA and GEO verified the oncogenic characteristics of PRMT5 and MCM7 in CRC. In summary, our data provided new insight into PRMT5 and MCM7 as potential diagnostic and therapeutic targets in CRC.22.1A total of 99 CRC and 77 adjacent normal samples were collected from the National Engineering Center for Biochip at Shanghai (SBC). All CRC tissues were diagnosed histologically. This study was permitted and approved by the Ethics Committee at Zhejiang University.2.2The human CRC cell lines RKO, HT29, HCT8 and HCT116 and the human embryonic kidney cell line HEK293T were obtained from the American Type Culture Collection . HT29, HCT8 and HCT116 cells were maintained in RPMI‐1640 medium  supplemented with 10% foetal bovine serum (Gibco), while RKO and HEK293T cells were grown in Dulbecco's modified Eagle's medium (Gibco) containing 10% foetal bovine serum.2.3The cDNA encoding the full‐length PRMT5 open reading frame (ORF) was amplified and engineered into the lentiviral vector pHBLV with a luciferin reporter; the sequences of the primers used for the generation of constructs are listed in Table 2.4The full‐length ORF or sequences of various truncation mutants of PRMT5 or MCM7 were amplified and inserted into the pcDNA3.1‐Myc or pcDNA3.1‐FLAG vector. The sequences of the primers used for the generation of constructs are listed in Table 2.5Total RNA was isolated using Trizol (Invitrogen) and subsequently reverse transcribed into cDNA using PrimeScript RT reagent kit . Then qPCR was performed with SYBR green  and normalized to GAPDH according to manufacturer protocols. The sequences of the primers are listed in Table 2.65 cells in 100 μL of PBS) were injected into the tail veins of 5‐ to 6‐week‐old BALB/c nude mice. Six weeks after injection, mice were anaesthetized with isoflurane (Sigma) and intraperitoneally injected with D‐Luciferin substrate  prior to analysis of bioluminescence signals using an in vivo imaging system (IVIS) Spectrum System . After mice were sacrificed, lungs and brains were surgically excised and processed for staining with haematoxylin and eosin (H&E). All mouse studies and procedures were conducted in accordance with the guidelines established by the Animal Care and Use Committee of Zhejiang University.Control or PRMT5‐overexpressing HT29 cells  overnight at 4°C with gentle agitation. The precipitated samples were washed three times and further analysed by western blotting using the indicated antibodies.2.8t test was used for comparisons between two groups, and one‐way ANOVA was used for comparisons among three or more groups. All statistical analyses were performed using SPSS 17.0 software  or GraphPad Prism 7.0 software . P < .05 was considered statistically significant.All results are presented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) values. Student's 33.1E‐cadherin levels, whereas it increased Vimentin and c‐Myc expression levels  were transfected with control siRNA or siRNA targeting PRMT5 (si‐PRMT5). The efficiency of PRMT5 silencing was confirmed by western blotting Figure , and celTo validate the above results in vivo, we implanted luciferase‐labelled HT29 cells with or without PRMT5 overexpression into nude mice via the lateral tail vein to examine the effect of PRMT5 on metastasis. Metastasis formation was monitored by bioluminescence imaging with an IVIS system. Consistent with the role of PRMT5 in vitro, we found that overexpression of PRMT5 significantly promoted distant metastasis to the lungs and brain Figure . After 63.2E‐cadherin was significantly induced upon loss of MCM7, whereas the expression levels of Vimentin and c‐Myc were reduced  analysis to identify the potential PRMT5‐interacting proteins in HT29‐PRMT5 cells. MCM7, WDR77 and other proteins were identified as potential binding partners of PRMT5 Table . We nextd Figure . Taken t3.3R = .420, P < .001), further suggesting the correlation and interaction between PRMT5 and MCM7 in CRC.To detect PRMT5 expression in human CRC samples and determine its potential association with clinical outcomes, we carried out IHC assays and used Image‐Pro Plus 6.0 software to analyse PRMT5 expression in 99 CRC and 77 adjacent non‐cancerous tissues. Importantly, PRMT5 protein levels were found to be significantly higher in CRC samples than in normal control tissues Figure . ConsistP = .026, Figure P = .021, Figure To further evaluate the prognostic value of PRMT5 in CRC, the patients were divided into the low and high expression groups, and overall survival analysis was conducted. The Kaplan‐Meier curves showed that compared with patients with low PRMT5 levels, patients with high PRMT5 levels were characterized by poor overall survival in CRC ,GSE24514 (15 adjacent control and 34 CRC tissues),GSE22598 (17 paired tissues),GSE31737 (40 paired tissues),GSE89076 (39 adjacent control and 41 CRC tissues),GSE20842 (65 paired tissues),GSE44861 (55 adjacent control and 56 CRC tissues),GSE33113 (6 adjacent control and 90 CRC tissues),GSE60331 (19 adjacent control and 31 CRC tissues),GSE89287 (17 adjacent control and 54 CRC tissues),GSE23878 (24 adjacent control and 35 CRC tissues)GSE50421 (25 adjacent control and 24 CRC tissues).PRMT5 mRNA levels were significantly increased in CRC compared with non‐cancerous tissues in the TCGA (P < .001), GSE21510 (P < .001), GSE24514 (P < .001), GSE22598 (P < .001), GSE31737 (P < .001), GSE89076 (P < .001), GSE20842 (P < .01), GSE44861 (P < .05), and GSE33113 (P < .001) datasets. Additionally, we analysed MCM7 mRNA expression in the TCGA and GEO data sets. As shown in Figure MCM7 mRNA levels were remarkably increased in CRC compared with non‐cancerous tissues in the TCGA (P < .001), GSE21510 (P < .001), GSE24514 (P < .001), GSE22598 (P < .001), GSE31737 (P < .001), GSE60331 (P < .001), GSE89287 (P < .01), GSE23878 (P < .001) and GSE50421 (P < .001) data sets. Collectively, our data demonstrated that PRMT5 and MCM7 mRNA levels were high in CRC tissues in public data sets and might be involved in the pathogenesis of CRC.To validate the previous observations, we employed public data sets from TCGA and GEO to evaluate 4PRMT family proteins are a large class of enzymes that are well characterized as protein arginine methyltransferases. Based on patterns of arginine methylation, the nine mammalian PRMTs are classified into two major groups: type I and type II.The MCM family of proteins, which is highly conserved, was originally identified for its function in minichromosome maintenance in yeast. As components of the pre‐replicative complex (Pre‐RC), six subunits (MCM2‐7) interact to form the heterohexameric MCM complex during early G1 phase.Melicope ptelefolia (MP) hexane extract as an agent with anticancer therapeutic potential via its modulation of the expression of cancer‐associated genes. Importantly, it significantly attenuated MCM7 expression in CRC.As mentioned above, MCM7, known as a member of the MCM family, is a pivotal component of the MCM complex, in which, it allows the initiation of eukaryotic genome replication during G1 phase and elongation of DNA strands during S‐phase. Upregulated MCM7 expression is believed to be related to increased cell proliferation and tumour progression. A variety of MCM7‐binding partners have been identified to date. For instance, Sterner et alPRMT5 is known to form a complex with MEP50 and catalyse the methylation of arginine residues in histones and non‐histone proteins. PRMT5 methylation of histone H4R3 and H3R8 is generally correlated with transcriptional repression, whereas PRMT5‐catalyzed H3R2 methylation is generally correlated with transcriptional activation.PRMT5 and MCM7 mRNA in CRC. Additionally, we found that PRMT5 could be a prognostic predictor in CRC patients. Kaplan‐Meier survival analysis indicated that individuals with high PRMT5 expression showed poor clinical outcomes among total CRC patients and in the subgroup with a tumour size of ≤5 cm. Taken together, the results of this study demonstrated that PRMT5 promotes the malignant behaviour of colorectal cancer by interacting with MCM7. In conclusion, our study supports the targeting of PRMT5 and MCM7 as novel clinical intervention strategies in CRC.In this study, we investigated the oncogenic roles of PRMT5 and MCM7 in CRC. We revealed a new form of molecular interplay between PRMT5 and MCM7 in the pathogenesis of CRC. A direct association between the oncoprotein PRMT5 and MCM7 was identified. We found that PRMT5 promoted cell proliferation, migration and invasion in CRC in vitro and in vivo. To elucidate the molecular mechanisms, co‐IP and MS analysis were performed, and we identified a variety of potential PRMT5‐binding proteins, including WDR77 and MCM7. Our co‐IP results confirmed that PRMT5 directly interacted with the MCM7 protein. The fragment of MCM7 spanning amino acids 1‐248 was found to be required for its interaction with PRMT5. In addition, we evaluated the expression of PRMT5 and MCM7 in a large cohort of 99 CRC and 77 adjacent non‐cancerous tissues. IHC staining demonstrated that upregulation of PRMT5 and MCM7 frequently occurred in CRC tissues compared to controls. Our results also revealed a positive correlation between the PRMT5 and MCM7 protein levels. Bioinformatic analysis using TCGA and GEO data sets confirmed the elevated levels of All the authors declare no conflict of interest.Xiangwei Li: Data curation (lead); Formal analysis (lead); Methodology (lead); Writing‐review & editing . Xin Wang: Data curation ; Methodology ; Writing‐review & editing . Jiahui Zhao: Data curation ; Methodology ; Writing‐review & editing . Jian Wang: Conceptualization (lead); Funding acquisition (lead); Project administration ; Supervision ; Writing‐review & editing . Jingjing Wu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Writing‐original draft (lead); Writing‐review & editing .Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1‐S4Click here for additional data file.


PKC-ζ overexpression exacerbated Dox-induced cardiotoxicity. Conversely, knockdown of PKC-ζ by siRNA relieved Dox-induced cardiotoxicity. Similar results were observed when PKC-ζ enzyme activity was inhibited by its pseudosubstrate inhibitor, Myristoylated. PKC-ζ interacted with β-catenin and inhibited Wnt/β-catenin signaling pathway. Activation of Wnt/β-catenin signaling by LiCl protected against Dox-induced cardiotoxicity. The Wnt/β-catenin inhibitor XAV-939 aggravated Dox-caused decline of β-catenin and cardiomyocyte apoptosis and mitochondrial damage. Moreover, activation of Wnt/β-catenin suppressed aggravation of Dox-induced cardiotoxicity due to PKC-ζ overexpression. Taken together, our study revealed that inhibition of PKC-ζ activity was a potential cardioprotective approach to preventing Dox-induced cardiac injury.Doxorubicin (Dox) is a chemotherapeutic drug used to treat a wide range of cancers, but its clinical application is limited due to its cardiotoxicity. Protein kinase C-ζ (PKC-ζ) is a serine/threonine kinase belonging to atypical protein kinase C (PKC) subfamily, and is activated by its phosphorylation. We and others have reported that PKC-ζ induced cardiac hypertrophy by activating the inflammatory signaling pathway. This study focused on whether PKC-ζ played an important role in Dox-induced cardiotoxicity. We found that PKC-ζ phosphorylation was increased by Dox treatment  Doxorubicin (Dox) is a widely used chemotherapeutic agent for treatment of leukemia, lymphoma, neuroblastoma, and other human cancers . HoweverProtein kinase C-ζ (PKC-ζ) is a serine/threonine kinase belonging to atypical protein kinase C (PKC) subfamily . PKC-ζ aWnt/β-catenin is a highly conserved Wnt signaling pathway branch. As an important transcriptional co-activator, β-catenin protein dynamic balance plays a key role in maintaining cell homeostasis . Wnt/β-cThis study explored the role of PKC-ζ in Dox-induced cardiotoxicity and underlying mechanisms. Results of the current study indicated that PKC-ζ acted as upstream suppressor of Wnt/β-catenin signaling and aggravated Dox-induced cardiac injury. PKC-ζ-based intervention was a potential strategy for alleviating Dox-induced cardiotoxicity.Dox (purity 99.36%) was purchased from Target Molecule Corp (United States). Small-interfering RNA (siRNA) of PKC-ζ was purchased from Genema . The PKC-ζ pseudo-substrate inhibitor Myristoylated was purchased from Santa Cruz (United States). XAV-939 was purchased from Selleck Chemicals (United States). LiCl was purchased from Sigma (United States).Guide for the Care and Use of Laboratory Animals  and approved by the Research Ethics Committee of Sun Yat-sen University. Male Sprague–Dawley (SD) rats  were purchased from the Experimental Animal Center of Sun Yat-sen University . Animals were randomly divided into two groups, model and control groups, and each group contained 6 rats. Rats in the model group were intraperitoneally administered with Dox on the 1st, 5th, and 9th day at a dosage of 5 mg/kg. Rats in the control group were intraperitoneally administered with the same dosage of normal saline (NS).Animal experimental procedures were undertaken in compliance with the The day after the final administration of Dox, SD rats were anesthetized with 3% isoflurane, and echocardiographic measurements were taken using Technos MPX ultrasound system  of two-dimensional-guided M-mode echocardiography based on previous studies . CardiacHeart tissues of SD rats (1–3 days old) were isolated based on a previously described protocol  to obtaiProteins of cardiomyocytes or heart tissues were extracted using RIPA lysis buffer. Equal amounts of 30- or 35-μg proteins were used to undertake immunoblotting experiment as previously described . PrimaryDNA sequence of pEGFP-N3-Flag-PKC-ζ plasmid was confirmed by Sangon Biotech Co. Ltd. (Shanghai). Plasmid was transiently transfected into cardiomyocytes based on the manufacturer’s instructions of Lipo 2000. The siRNA sequence of PKC-ζ included sense 5′-GCAAGCUGCUUGUCCAUAATT-3′ and antisense 5′-UUAUGGACAAGCAGCUUGCTT-3’. Cardiomyocytes were transfected with siRNA or negative control based on RNAiMAX transfection reagent instructions for use. All experiments were conducted in 48–72 h after transfection.Cardiomyocytes were seeded into 48-well plates. After corresponding treatments, cells were washed thrice with PBS and then incubated with 10 nM tetra-methylrhodamine ethyl ester (TMRE)  for 30 min followed by 10 μg/ml Hoechst 33,42 staining  for 10 min at 37°C. Mitochondrial membrane potential and nuclear condensation were detected using EVOS FL Auto .After washing with PBS, cardiomyocytes were fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton X-100 for 10 min at room temperature; cardiomyocytes were then incubated with 1 μM MitoTracker Red  for 30 min followed by 10 μg/ml Hoechst 33342 staining for 10 min. Mitochondrial morphology was detected using an ultra-high-resolution laser scanning microscope .p-PKC-ζ , or Troponin T  were used to incubate cells overnight at 4°C. Next, cardiomyocytes were incubated with Alexa Fluor 488- or 594-conjugated anti-rabbit or mouse IgG (H + L) secondary antibody  at room temperature for 1 h. After nuclear staining by DAPI for 10 min, cardiomyocytes were detected using an ultra-high-resolution laser scanning microscope .Cardiomyocytes were seeded into coverglass bottom dishes. After washing thrice with PBS, cells were fixed with 4% paraformaldehyde (20 min) and permeabilized using 0.3% Triton X-100 (10 min). Then, cells were washed with PBS for another three times and were blocked with 10% goat serum at room temperature for 1 h. Antibodies against PKC-ζ (diluted 1:50), β-catenin (diluted 1:100), A total of 300–400 μg of protein was respectively incubated with anti-β-catenin (diluted 1:100) or anti-PKC-ζ antibody (diluted 1:50) and IgG antibody  overnight at 4°C, followed by incubation with 20 μl of protein A/G beads  for 4 h at 4°C. Then, beads were washed with IP Lysis buffer and diluted in loading buffer. The interactive protein was assayed by immunoblotting.t-tests. One-way ANOVA with Bonferroni post hoc tests were undertaken for multiple group comparisons. p < 0.05 was considered statistically significant.Data are presented as mean ± SEM. Statistical analyses between two groups were done using Student’s in vivo and in vitro. SD rats were intraperitoneally administered with Dox at a dosage of 5 mg/kg body weight on the 1st, 5th, and 9th day, leading to a cumulative dosage of 15 mg/kg body weight. The echocardiographic analysis revealed decreased ejection fraction (EF%), fractional shortening (FS%), and left ventricular posterior wall thickness (LVPW) in Dox-treated group rats compared with those of control group rats , which are regulated by calcium, DAG, and phospholipids; novel PKC isoforms , which are activated by DAG and phospholipids; and atypical PKC isoforms (ζ and λ or ι), whose activation depends on protein–protein interaction and lipids such as ceramide rather than calcium and DAG . Finding2O2-induced H9C2 cells against apoptosis by upregulating the Wnt/β-catenin pathway (via inhibiting Wnt/β-catenin (2O2-induced apoptosis in endothelial cells (in vivo and in vitro. Activating Wnt/β-catenin signaling by LiCl alleviated Dox-induced cardiomyopathy (Evidence has shown that the Wnt/β-catenin pathway is implicated in cardiac differentiation , apoptos pathway . MiR-148-catenin . β-catenal cells . Wnt/β‐cal cells . In addimyopathy , while Kmyopathy . Wnt/β‐cmyopathy . Severalmyopathy . Victorimyopathy . We postIn summary, the current study established that loss of function of PKC-ζ is a potential therapeutic approach to alleviate Dox-induced cardiac injury. In addition, the current study indicated that the deleterious effect of PKC-ζ is partly elucidated by inhibiting Wnt/β-catenin signaling. Further studies on cardiac-specific overexpression or knockdown of PKC-ζ in animal models are needed to further validate all the observed cellular findings in the current study. Moreover, specific regulatory mechanisms underlying PKC-ζ regulation of β-catenin should be explored further.


CP) of cold-mix resin and cold-mix resin asphalts exhibit a sudden change between −20 °C and 40 °C. In resin asphalt mixtures, cold-mix resin forms the network structure skeleton whereas the asphalt distributed in the form of tiny particles. The dosage of respective component has a significant effect on the tensile strength and elongation at break of cold-mix resin. Compared with hot-mix epoxy asphalt mixtures, cold-mix resin mixtures exhibit comparable water stability and high and low-temperature performance, as well as greater fatigue life.To improve the safety of orthotropic steel bridge decks and the construction efficiency of bridge deck pavement by enhancing the performance of pavement materials, a new-generation, high-performance cold-mix resin was prepared by carrying out the combination of micro-characteristic analysis and performance test. Meanwhile, the pavement performance and fatigue performance of high-performance cold-mix resin mixtures and hot-mix epoxy saphalt mixtures as a control group were studied experimentally. The results show that different kinds of epoxy resins show bisphenol structure in essence. The curing exothermic peak temperature of the cold-mix resin increases with the heating rate. Both the specific heat capacity (△ Orthotropic steel bridges have advantages, such as being light, able to span large distances, a high bearing capacity and convenient construction ,2,3,4. ANowadays, epoxy asphalt materials widely employed in China mainly includes hot-mix epoxy asphalt (HMA) and warm-mix epoxy asphalt (WMA). In order to obtain and maintain the fluidity of hot-mix epoxy asphalt during construction, a large amount of heat was supplied by machinery through energy consumption . With thDespite the fact that the incorporation of epoxy resin brought excellent performance for bridge deck paving ,20,21, mOn the basis of the existing literature, this research aims to prepare a novel material with short curing time for bridge deck pavement. In this paper, three types of widely applied epoxy resins are tested by infrared (IR) spectrum to analyze specific components and the physical properties as well as the micro-characteristics of resin and resin asphalts are analyzed. By adjusting the proportion of respective component in resin, orthogonal tests of tensile fracture are designated and the optimal dosage of respective component is determined. Ultimately, a novel paving material is identified, namely, high-performance cold-mix resin mixtures, which exhibits equivalent pavement performance and superior fatigue performance compared with hot-mix epoxy asphalt mixtures.Three typical epoxy resins provided by Nanjing Chengyou Resin Co. Ltd., Nanjing, China were prepared, including Dow resin, TY-RA resin and YC-YN resin. E-51 is a kind of epoxy resin with Bisphenol-A type structure, which is the preferred base materials for preparing cold-mix resins ; low-molKBr tablet method, the samples were dissolved using anhydrous ethanol solution and then coated onto KBr tablets. After the anhydrous ethanol solution was heated and evaporated, IR spectra of the coated materials were determined within the scanning range of 400 cm−1 to 4000 cm−1.According to the test method proposed in GB/T 6040-2002 , the infThe differential scanning calorimetry (DSC)  was used to measure the heat released from curing of high-performance cold-mix resin as well as the glass transition temperature of the cured high-performance cold-mix resin and high-performance cold-mix resin asphalts . With thScanning electron microscopy (SEM)  is an observation method somewhere between transmission electron microscopy (TEM) and optical microscopy. The resolution of the SEM used in this paper reaches 1 nm and the microscope can be continuously adjusted at the magnification of 300,000 times and above. The cured high-performance cold-mix resin samples were brittle fractured in liquid nitrogen, then tetrahydrofuran (THF) was added and heated together at 65 °C for 15 h. After the samples were dried at room temperature, the cross-section of the samples were sprayed with gold, then the samples were placed in the fixture of the instrument to observe the microscopic appearance of the cross-section. In order to better observe the microscopic appearance of high-performance cold-mix resin mixed with asphalts, the corresponding test was carried out on a laser scanning confocal microscope (LSCM)  . After tThe fracture tensile test is regarded as an important method to evaluate the basic mechanical properties of high-performance cold-mix resin. In accordance with the designed orthogonal experiments, a certain amount of E-51, 651, ETBN, ETPEG, KH-550 and DMP-30 were mixed and stirred more than 180 s in each set of tests. The mixtures were poured into the mold and then placed in an environmental box cured at 25 °C for 12 h. Afterwards, the samples were cured at 60 °C in an oven for 16 h, then fracture tensile tests were carried out on QJ211S  test machine to measure the tensile strength and percentage of breaking elongation of high-performance cold-mix resin. The tensile rate in test was set at 10 mm/min.Through the orthogonal experiment designed in fracture tensile test and further analysis, the best ratio indicates that the copies of E-51, 651, ETBN, ETPEG, KH-550 and DMP-30 in high-performance cold-mix resin were 50, 55, 50, 6, 3 and 2, respectively. The coarse and fine aggregates of the same type as those applied in epoxy asphalt concrete pavement of steel bridge deck were selected. The coarse and fine aggregates as well as mineral powder were rolled from basalt and limestone, respectively. In addition, polyester staple fiber accounting for 0.1% of the mass of mineral raw materials was also employed. Meanwhile, hot-mix epoxy asphalt mixtures were designed for comparison with high-performance cold-mix resin mixtures. The coarse and fine aggregates applied in hot-mix epoxy asphalt mixtures are the same as those applied in high-performance cold-mix resin mixtures. The hot-mix epoxy asphalt mixtures were prepared in accordance with the following procedures: the modifier, curing agent and base asphalt were heated to 80 °C, 60 °C and 170 °C, respectively, then the modifier and curing agent were mixed according to the weight ratio of 1:2. The obtained mixtures were mixed and stirred with epoxy resin in a 1:1 weight ratio, followed by the addition of aggregates which were heated to 185 °C. As a significant index to evaluate the pavement performance of road materials, water stability is mainly applied to measure the deterioration of the performance of mixtures under hydrodynamic pressure. In this section, the widely applied water immersion Marshall test and freeze-thaw splitting test were performed on high-performance cold-mix resin mixtures and hot-mix epoxy asphalt mixtures by following the standard test procedure in in JTG E20-2011  and the Under the combined effect of high temperature ambient and vehicle load, the road materials would soften and then deform. In addition, the combined action of low temperature ambient and vehicle load would cause pavement cracking and finally, induce the structural damage under adverse environmental conditions such as rain and snow erosion. Therefore, the high and low stabilities of the designed mixtures is also an important part of the road performance research. In view of this, the rutting test and beam bending test confirming to the standard test procedure in JTG E20-2011  were carThe four-point bending fatigue tests referred to the method suggested in JTG E20-2011 . Corresp−1 and the vibration absorption peak of the benzene ring skeleton in the bisphenol-A structure is found at 1607 cm−1. Some epoxy resins are plasticized, toughened and modified by mixing with asphalt. The broad band at 3285 cm−1 shows the vibration absorption peak of hydroxyl groups or the double-frequency absorption induced by stretching vibration of N-H and carbonyl. There are many impurities represented by peaks between 900 cm−1 and 1300 cm−1 in the fingerprint region.The IR spectrum of components A and B in three typical epoxy resins, namely Dow epoxy resin, TY-RA epoxy resin and YC-YN epoxy resin, are shown in −1 to 3100 cm−1, representing the vibration absorption peak of hydroxyl. By comparing the results, the peak is found to be ascribed to Hardener XU-HY 943, mainly including  dimethyldipropylamines and polyamides. The component B in TY-RA epoxy resin presents characteristic peaks of polyamides, namely, strong peaks at 1640 cm−1 and 1550 cm−1. The comparison demonstrate that the peaks are mainly attributed to Paramul ERO-SB compound, which mainly comprises polyamides. YC-YN epoxy resin are similar to TY-RA epoxy resin in component B, which is a room-temperature curing agent containing polyamides.A comparison with the data from spectrum library shows that Dow epoxy resin is very similar to bisphenol-A resin in terms of component A, both mainly containing DER332. Such resin show epoxy equivalent of 172 to 176 (g/eq) and an epoxy value of 0.56 to 0.58; and the main ingredient of component A in TY-RA epoxy resin is DER 324, as well as doped asphalts, whereas the main ingredient of component A in YC-YN epoxy resin is EPON 828 as well as doped asphalts. Component B in epoxy resins mainly includes various curing agents and curing of epoxy resins at room temperature generally relies on an amine curing agent, in particular, polyamides. Based on analysis of IR absorption spectra, the component B in Dow epoxy resin shows an obvious band near 3000 cmBased on the above analysis, the results of IR spectrum analysis of three typical epoxy resins are list in Tg) plays a significant role in the complex low-temperature behavior of materials [Tg) of completely cured high-performance cold-mix resin and high-performance cold-mix resin asphalts. It can be intuitively seen that the specific heat capacity (ΔCP) of high-performance cold-mix resin and high-performance cold-mix resin asphalts exhibit a sudden change between −20 °C and 40 °C and the corresponding glass transition temperature are both about 12.4 °C. The low glass transition temperature indicates that high-performance cold-mix resin and high-performance cold-mix resin asphalts in low temperature conditions exhibit good deformation capacity. Generally, the glass transition temperature of pure asphalt is about −20 °C, which indicates that pure asphalt shows good viscoelasticity.The glass transition temperature  dimethyldipropylamines and polyamides, while that in TY-RA resins is the Paramul ERO-SB compound, mainly consisting of polyamides. Moreover, the room-temperature curing agent containing polyamides is mainly found in Component B of YC-YN resins.CP) in the range of −20 °C to 40 °C and the corresponding glass transition temperature is about 12.4 °C, presenting a low Tg, that is, a high denaturation capacity can be retained even at this low temperature.Through DSC, different exothermic peaks appear at different heating rates of 5, 10, 15 and 20 °C/min after curing cold-mix epoxy asphalt mixtures and the faster the rate of heating, the higher the peak temperatures . Both cold-mix resin and cold-mix resin asphalt show a sudden change in specific heat capacity (△The results of orthogonal fracture tensile test and range analysis illustrate that the effect of resin matrix, ETBN, 651 polyamide curing agent and KH-550 coupling agent content on tensile strength of high-performance cold-mix resin is greater than that of ETPEG diluter and DMP-30 accelerator content on tensile strength of high-performance cold-mix resin. In terms of the elongation at break of high-performance cold-mix resin, the dosage of E51 resin generates the greatest effect whereas the ETPEG generates the least effect.Pavement performance tests show that high-performance cold-mix resin mixtures exhibits comparable residual Marshall stability and freeze-thaw splitting tensile strength ratio as well as flexural tensile strength and ultimate flexural tensile strain at low temperature, whereas larger Marshall stability and splitting tensile strength as well as greater dynamic stability at high temperature, compared with hot-mix epoxy asphalt mixtures. The four-point bending tests show that, at strain of 400 με, the fatigue life of high-performance cold-mix resin mixtures is much greater than that of hot-mix epoxy asphalt mixtures.


Background: Previous studies have demonstrated that long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explores the use of MEG3 in skull defects bone repairing. Methods: Endogenous expression of MEG3 during BMSCs osteogenic differentiation was detected by quantitative real-time polymerase chain reaction (qPCR). MEG3 was knockdown in BMSCs by lentiviral transduction. The proliferation, osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs were assessed by Cell Counting Kit-8 (CCK-8) assay, qPCR, alizarin red and alkaline phosphatase staining. Western blot was used to detect β-catenin expression in MEG3 knockdown BMSCs. Dickkopf 1 (DKK1) was used to block wnt/β-catenin pathway. The osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs after wnt/β-catenin inhibition were assessed by qPCR, alizarin red and alkaline phosphatase staining. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model. Micro-computed tomography(micro-CT), hematoxylin and eosin staining and immunohistochemistry were performed to evaluate the bone repairing. Results: Endogenous expression of MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed that MEG3 knockdown could activate Wnt/β-catenin signaling pathway in BMSCs. Wnt/β-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using poly -mesoporous bioactive glass (PHMG) scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerate bone repairing in a critical-sized skull defects rat model. Conclusions: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects. In the clinic, skull defects caused by trauma, severe infection, tumor resection and decompressive craniectomy due to refractory high intracranial pressure are very common in neurosurgery. Although autologous bone transplantation, allogeneic bone transplantation, titanium mesh or polyether-ether-ketone (PEEK) cranioplasty were widely used in the clinical treatment of skull defects, complications such as donor site morbidity, bone resorption/loosening graft infection, implant exposure and high costs remain unresolved ,2,3. BonMaternally expressed gene 3 (MEG3) is a maternally expressed long non-coding RNA, and increasing evidence has revealed that MEG3 emerged as a key regulator in the process of development and tumorigenesis ,8. We haWnt/β-catenin signaling is a crucial pathway in the skeleton controlling osteoblast differentiation and bone formation . With wnIn this study, we found the endogenous expression of MEG3 increased during the process of osteogenic differentiation. Based on previous studies and our results, we hypothesize that MEG3 may participate in osteogenic differentiation of BMSCs and bone regeneration. The function of MEG3 during osteogenic differentiation of BMSCs was demonstrated by assessing the expression levels of osteogenic genes, alkaline phosphatase activity and calcium deposition. Notably, a further mechanism study revealed that the pro-osteogenic effects of MEG3 may be partially attributed to wnt/β-catenin signaling pathway. A composite poly -mesoporous bioactive glass (MBG) scaffold (PHMG), composed of biodegradable PHBHHx and MBG, was selected as the vehicle in this study, owing to the advantages of its good biocompatibility, favorable loading behavior of its mesoporous structure and so on. Our previous studies have shown that composite PHMG scaffolds have no cytotoxicity and improved cellular affinity; furthermore, PHMG possesses hierarchical mesoporous structure with a comparatively large specific surface area for cellular adhesion and spread . PHMG sc5 cells/mL was used in the cellular tests. Recombinant DKK1 was purchased from PeproTech . In accordance with a previous study, the applied concentration of DKK1 was 0.5 μg/mL [Human bone marrow stromal cells (hBMSCs) were obtained from four donors who gave their written informed consent. Briefly, marrow was extracted from the femoral midshaft and then suspended in minimum essential medium containing 10% fetal bovine serum , 100 U/mL penicillin and 100 mg/L streptomycin. Subsequently, the non-adherent cells were discarded; the adherent cells converged to 80–90% confluence and were then replated as passage one (P1) cells. P3 cells were used for experiments. A density of 1 × 10.5 μg/mL ,28.Lentivirus knockdown MEG3 particles and lentiviral RFP particles were described as previously . In brieTo assess the effect of MEG3 downregulation on the proliferation of hBMSCs, the cells were seeded into a 96-well plate (5000/well) and allowed to adhere for 24 h. After 24 h, the medium was removed, and the cells were treated with 10% CCK-8  in 100 μL low-sugar Dulbecco’s modified Eagle’s medium (L-DMEM) without fetal bovine serum (FBS) for 2 h at 37 °C. Absorbance at 450 nm, which is directly proportional to cell proliferation, was measured using a microplate reader .4/cm2, and incubated for 48 h at 37 °C under 5% CO2. The cells were subsequently cultured in osteogenic induction medium . The cells were maintained by the addition of fresh osteogenic induction medium every 3 days.BMSCs were cultured in growth medium  and 100 IU/mL penicillin/streptomycin) in 6- or 12-well cell culture plates , at a density of 3 × 10For the measurement of ALP activity, cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer , and the lysate (10 μL) was incubated with 90 μL fresh solution containing p-nitrophenyl phosphate substrate at 37 °C for 30 min. The reaction was stopped by the addition of 0.5N NaOH (100 μL), and the absorbance was measured at 405 nm using a microplate reader . The total protein concentration was measured using a BCA protein assay kit . The relative ALP activity is expressed as the percentage change in optical density (OD) per unit time per milligram protein: (OD/15 min/mg protein) × 100.After the induction of osteogenic differentiation, mineral deposition was assessed by ARS (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde  for 15 min at room temperature and then washed with distilled water. A 1% solution of alizarin red was added and incubated for 30 min at room temperature, followed by rinsing with distilled water. The solution was collected, and 200 μL were plated on 96-well plates, which were read at 560 nm using a microplate reader . The readings were normalized to the total protein concentration.−ΔΔCt method.Total cellular RNA was isolated using RNAiso reagent  and quantified by measuring the absorbance at 260 nm . Total RNA (≤1000 ng) was reverse-transcribed into cDNA in a reaction volume of 20 μL using the Double-Strand cDNA Synthesis Kit . One microliter of cDNA was used as the template for the qPCR reaction. All gene transcripts were quantified by qPCR using the Power SYBR Green PCR Master Mix (Takara) on the ABI StepOnePlus System . The mRNAs of the target genes and GAPDH were quantified in separate tubes. All primers were synthesized by Sangon Biotech . The primer sequences used are shown in Cells were lysed in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane . After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to β-actin  or β-catenin . Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  was applied as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent . Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system .4 cells/scaffold. After 4 h, 100 μL of culture medium was carefully added to the base of the culture plate until the scaffold was covered with sufficient culture medium.Prior to seeding cells, the prefabricated PHMG scaffolds were sterilized using gamma irradiation. Cell suspension (100 μL) was added to four groups at a density of 1 × 10Animal experiments were approved by the Research Ethics Committee of the Shanghai General Hospital and performed in accordance with the Care and Use of Laboratory Animals protocols. Briefly, mature Sprague Dawley (SD) male rats (mean body weight 250–300 g) were provided with sterilized food and water and housed in a barrier facility with a 12-h light/dark cycle. These rats were randomly divided into three groups, each containing six rats: PHMG, PHMG + sh-Ctrl and PHMG + sh-MEG3 group. For the surgical procedure, as previously described, the animals were anesthetized by intraperitoneal injection of chloral hydrate  and all operations were performed under sterile conditions. A 1.5-cm sagittal incision was made in the scalp and the calvarium was exposed by blunt dissection. Two critical-sized calvarial defects with a bilateral diameter of 5 mm were created using a dental trephine, and the scaffolds were then implanted into the defects. Following the operation, the animals received intramuscular antibiotic injections, were allowed free access to food and water and were monitored daily for potential complications. Eight weeks after the operation, the rats were killed by an overdose of anesthetics and their craniums were harvested and fixed in a 4% paraformaldehyde solution buffered with 0.1 M phosphate solution (pH 7.2) overnight before further analysis.®  were scanned at a resolution of 9 μm. After 3D reconstruction, the bone mineral density (BMD) and bone volume fraction ) in the defect regions were used to calculate new bone formation using the auxiliary software of the mCT-80 system.All the harvested specimens were examined using the mCT-80 system to evaluate new bone formation within the defect region. Briefly, the undecalcified samples were scanned at a resolution of 18 μm and decalcified samples perfused with MicrofilOne part of calvarias was decalcified in 10% EDTA for 14 days, dehydrated with graded ethanol solutions, embedded in paraffin and sectioned at 5 μm at the central area of the defect. Sections were stained with hematoxylin and eosin (HE) to observe new bone formation.The other part of each cranium was decalcified for approximately 2 weeks, dehydrated using a graded alcohol series, embedded in paraffin and sectioned into 5 μm sections. Osteocalcin (OCN) IHC was performed to evaluate osteogenesis in specimens. Briefly, deparaffinized sections were washed with PBS, treated with antigen retrieval and blocked with IgG for 30 min. OCN at a 1:200 dilution was incubated at 4 °C overnight. Then the biotinylated secondary antibody and ABC complex were applied and DAB substrate was used to stain the section. The sections were stained with hematoxylin and the results were analyzed with a light microscope.t-test when comparing two groups and one-way ANOVA followed by Bonferroni’s post hoc test when comparing more than two groups. p < 0.05 was considered to indicate statistical significance.Statistical analysis was performed using SPSS 17.0 software . All experiments were performed at least in triplicate, and the data are presented as means ± standard deviation. Statistical significance was determined using a two-tailed Student’s p < 0.05, To determine whether MEG3 was involved in osteogenic differentiation of BMSCs, we examined the expression MEG3 in BMSCs at day 0, 7 and 14 after osteoblastic induction. Compared with undifferentiated BMSCs, the expression of MEG3 was significantly increased at day 7 and 14 during the process of osteogenic differentiation  and osteocalcin (OCN) were detected by qPCR. qPCR analysis revealed that ALP, RUNX2 and OCN mRNA levels were significantly higher in sh-Meg3 group at day 7 and 14 than in blank group and sh-Ctrl group (p < 0.05, p < 0.05, We evaluated ALP activity, an early marker of osteogenesis, at day 14 during osteogenic differentiation. Compared with the blank group and sh-Ctrl group, higher ALP activity was observed in sh-Meg3 group (p < 0.05, Wnt/β-catenin signaling pathway plays an important role in the osteogenic differentiation of BMSCs. To gain insights into the mechanism by which MEG3 regulates osteogenic differentiation of BMSCs, the expression changes of β-catenin in BMSCs were performed. Western blot results showed that the protein level of β-catenin increased in the sh-Meg3 group when compared with sh-Ctrl group (p < 0.05, To verify the relevance of the Wnt/β-catenin pathway and MEG3 knockdown, we evaluated the expression of β-catenin between sh-Ctrl group and sh-Meg3 group treated with or without DKK1. Western blot results showed that MEG3 knockdown resulted in an increase of β-catenin in sh-Meg3 group compared to the sh-Ctrl group, and the upregulation of β-catenin could inhibit by DKK1 (p < 0.05, Next, we determined whether the osteogenesis effect of MEG3 knockdown on BMSCs was mediated via the Wnt/β-catenin pathway. After being treated with or without DKK1, osteo-specific genes of OCN and Runx2 were detected by qPCR in sh-Ctrl group and sh-Meg3 group. Compared with the sh-Ctrl group, silencing of MEG3 increased the expression of OCN and Runx2 in sh-Meg3 group, whereas the increasing of expression of OCN and Runx2 could blocked by DKK1 in sh-Meg3 group (p < 0.05, Moreover, there was also less matrix mineralization at day 28 of osteogenic differentiation in the sh-Meg3 group treated with DKK1than in sh-Meg3 group C,D. Bloc3, and there was a significant difference between the PHMG group and PHMG + sh-Meg3 groups (p < 0.05, p < 0.05, To verify the pro-osteogenesis effect of MEG3 in vivo, PHMG adherent with MEG3 knockdown BMSCs was implanted in a critical-sized skull defects of rat model. The 3D morphology and 2D slice images of the newly-formed calvarial bones of each group at week 8 are reconstructed by micro-CT (HE staining clearly showed that barely any new bone formation was found in the PHMG group (In this study, we found the endogenous expression of MEG3 was upregulated during osteogenesis in BMSCs. Furthermore, downregulation of MEG3 could promote the osteogenic differentiation of BMSCs. Moreover, we showed that decreasing the expression of MEG3 in BMSCs could markedly accelerate bone repairing with upregulated bone mineral density, bone volume and increased new bone generation in rat critical-sized skull defects model. Mechanistically, we found that silencing of MEG3 could promote osteogenic differentiation of BMSCs via the Wnt/β-catenin signaling pathway . To our BMSCs, combined with biomedical materials, hold great promise for regenerative medicine, especially for treating the unmet critical-sized bone defects in clinics. Therefore, effectively enhancing BMSCs osteogenic differentiation and BMSCs-mediated bone regeneration is crucial in bone tissue regeneration. Recent evidence has revealed that dysregulation of MEG3 is closely associated with bone or bone degenerative diseases. For example, Liu et al. found MEG3 is upregulated in non-union fracture bone  while MEWnt/β-catenin signaling is an essential pathway in the osteogenic differentiation of MSCs and bone maintenance. Wnt signaling results in cellular accumulation of Wnt/β-catenin, followed by nuclear translocation of β-catenin and activation of target genes ,33. A prThe present study has some limitations. First, although we indicated that downregulation of MEG3 mediates the Wnt/β-catenin signaling pathway to promote osteogenic differentiation of BMSCs, it is probably involved in the activation of other signaling pathways as well. We have previously demonstrated that MEG3 could interact with notch signaling to promote angiogenesis. Hence, other signaling pathways such as notch pathway could also play a role in MEG3 knockdown induced pro-osteogenic effect. Further work will be focused on these in our future research. Second, the mechanisms of MEG3 that interact with Wnt/β-catenin have not been clarified completely; research has shown that MEG3 could function as ceRNA or form RNA-DNA triplex structures to regulate gene expression and thus further studies are needed.Based on our data, we found that downregulation of MEG3 could promote osteogenic differentiation of BMSCs and bone repairing, partly through activation of the Wnt/β-catenin signaling pathway. MEG3-modified BMSCs can be used as a novel promising strategy for skull defects.