To examine the pyrolysis and combustion characteristics of epoxy asphalt, the heat and smoke release characteristics were analyzed via TG-MS and cone calorimeter tests, and the surface morphology of residual carbon after pyrolysis and combustion was observed via scanning electron microscopy. The results showed that the smoke produce rate of epoxy asphalt was high in the early stage, and then sharply decreased. Moreover, the total smoke produced was close to that of base asphalt, and the surface of residual carbon presented an irregular network structure, which was rough and loose, and had few holes, however most of them existed in the form of embedded nonpenetration. The heat and smoke release characteristics of epoxy asphalt showed that it is not a simple fusion of base asphalt and epoxy resin. Instead, they promote, interact with, and affect each other, and the influence of epoxy resin was greater than that of base asphalt. At present, asphalt pavement is widely used in highway construction in China, and various asphalt modification technologies have been developed. Among them, epoxy asphalt is used for constructing steel bridge surfaces and tunnel pavement because of its high strength, high temperature resistance, and fatigue resistance ,2,3. Epo6/N2 environment via molecular dynamics. Li et al. [Wu et al.  used thei et al.  studied i et al. ,24 studii et al.  used AmsAccording to the composition characteristics of epoxy asphalt, the heat and smoke release characteristics of epoxy asphalt are analyzed via TG-MS and cone calorimeter tests, and the surface morphology of residual carbon after pyrolysis and combustion can be observed via scanning electron microscopy (SEM), which provides a theoretical basis for further analysis of the pyrolysis and combustion characteristics of epoxy asphalt.The base asphalt adopts Shell Pen 70, and the main performance indexes are listed in (1)TG-MSIn this study, the pyrolysis characteristics of base asphalt, epoxy resin, and epoxy asphalt were examined using the Japanese physio-thermo plus EV2/thermo mass photo TG-MS technology under a 70 eV electron ionization (EI) source. The heating range of TG ranged from 25 to 800 °C and the heating rate was 10 °C/min. The thermogravimetric analyzer and mass spectrometer were used simultaneously, and the scanning mode was ion scanning, simulating air atmosphere.(2)Cone Calorimeter Test2 of thermal radiation. The sample specification was 100 × 100 × 3 mm3.The cone calorimeter test is one of the most commonly used test instruments to study the combustion performance of materials. It is capable of examining time to ignition (TTI), HRR, THR, SPR, TSR, and gas parameters under different oxygen mole fractions, and is widely used in the evaluation of the combustion performance of materials. In this test, the FTT0402 cone calorimeter manufactured by the Fire Testing Technology Company in the UK was used for the mesoscale combustion test. The tests conducted were in accordance with ISO 5660-1 and ASTM E1354 standards. Each sample was wrapped with aluminum foil paper and was tested under 50 kW/mTG and differential thermal gravity (DTG) of epoxy asphalt are shown in max1 is observed at 342.8 °C, and the weight loss is approximately 38.1%. In this stage, the weight loss mainly occurs due to the fracture and degradation of the network structure of epoxy resin, the large amount of pyrolysis weight loss of saturated components, and participation of some small molecules of volatile aromatic components during pyrolysis. The saturated fraction and aromatic fraction are decomposed into alkanes and cycloalkanes under high-temperature conditions. With the further increase in temperature, the alkyl side chain breaks to generate some short-chain alkanes and gaseous products [max2 is observed at 440.8 °C, and the weight loss is approximately 23.8%. This stage mainly involves the decomposition of compounds and colloids formed in the previous stage. At higher temperatures, some relatively stable chemical structures begin to undergo cracking reaction and generate a large number of free radicals and a large number of gaseous products. Moreover, dehydrogenation polymerization of residual substances generates more stable macromolecular substances. The temperature in the third stage ranges from 467.3 to 579.9 °C and the corresponding Tmax3 is observed at 510.9 °C, and the weight loss is approximately 33.7%. This stage is mainly caused by the further degradation of semicoke polymer formed in the first stage of epoxy resin and the pyrolysis of heavy components in asphalt. In this stage, the carbonized layer generated by alkyl side-chain dehydrogenation polymerization in the first and second stages begins to crack and burn, and the heavy substance, asphaltene, in asphalt begins to burn [It can be seen from the figure that the weight loss of epoxy asphalt mainly occurs at 232.1–579.9 °C. Moreover, the weight loss rate is 96.13% and the maximum weight loss rate is −6.093 %/min. According to the weight loss rate curve, the pyrolysis weight loss process of epoxy asphalt can be divided into three consecutive stages. The temperature in the first stage ranges from 232.1 to 387.9 °C and the corresponding Tproducts . The tem to burn .The pyrolysis process of epoxy asphalt shows obvious multi-stage characteristics, indicating that the pyrolysis reaction process of epoxy asphalt is very complex. This is mainly because the base asphalt and epoxy resin are complex mixtures, and each component has different thermal stability. During the heating process, they gradually start pyrolysis, combustion, absorb, or release different amounts of heat, resulting in the multi-stage characteristics of the DTG curve.According to the test results of the MS analysis, in the first stage of pyrolysis, the percentage of weight loss was the largest; however, the amount of volatile released was insufficient and mainly included carbon monoxide, water, carbon dioxide, hydrogen peroxide, acetaldehyde, and propane. This is because in the first stage, the fracture and degradation of the epoxy resin network structure occurred with large weight loss; however, the composition of epoxy resin was relatively uncomplicated and less volatile. The third stage comprised the most volatile components, because with the gradual increase in temperature, the analysis of the light component in the saturated component showed that the alkyl side chain broke, there were more high molecular weight components, and the generated coke began to burn and release more volatiles.2O with a mass charge ratio of 18, N2, CO, and C2H4 with a mass charge ratio of 28, and CO2, C2H4O, N2O, and C3H8 with a mass charge ratio of 44. Compared with the second stage, the overall ion current intensity in the third stage is larger, and C3H8O, C2H4O2, C2H8N2, and C2H4S with a mass charge ratio of 60 and SO2 with a mass charge ratio of 64 are also produced.It can be seen from The FTT0402 cone calorimeter was used for the mesoscale combustion test. The test results of different materials are listed in Under strong thermal radiation, all types of materials quickly burned. The HRR reached the peak rapidly after ignition, and then abruptly dropped. The insufficient combustion of the polymer generated a large amount of black smoke and had a pungent smell. Within 500 s, all materials burned. Among them, the residual carbon remaining after pyrolysis and combustion of epoxy resin burned the least, and the residual carbon rate was less than 1%, which is consistent with the test results of thermogravimetric analysis. It was speculated that most substances in the materials were decomposed into gas components. Although base asphalt and epoxy asphalt had some residual carbon after combustion, the residual carbon was relatively broken and did not form a complete carbon layer.The HRR and THR of different materials are shown in TTI determined the flammability of polymer under actual fire conditions. The ignition time of each material ranged between 31 and 36 s. Moreover, resin materials were easier to ignite, and it took 6–8 min for each material to burn completely until it self-extinguished.2 and 104.5 MJ/m2, respectively. The HRR curve presented sharp peaks. Although the pkHRR and THR of base asphalt were the lowest, that is, 328.8 kW/m2 and 50.5 MJ/m2, respectively, the combustion curve was relatively smooth, indicating that the epoxy resin combustion was more intense, and the base asphalt combustion was less intense.HRR and THR are important for estimating the combustion safety of polymers. Both these parameters represent the thermal feedback generated in the material combustion process. The larger the HRR and THR, the more intense the pyrolysis process; however, it also results in worsening the safety. According to the test results, the time for base asphalt, epoxy resin, and epoxy asphalt to reach the peak of HRR was 100, 70, and 60 s, respectively. It was easier for the resin materials to reach the combustion conditions, and the peak heat release rate (pkHRR) and THR of epoxy resin were the highest, reaching 1015.7 kW/m2 production rate (CO2P) of different materials are shown in SPR, TSR, CO production rate (COP), and CO2/m2, indicating that the introduction of base asphalt into epoxy resin in the form of an interpenetrating network reduced the smoke generation of resin to a certain extent.Overall, the trend of SPR was consistent with that of HRR. When the HRR reached the peak, the SPR was the highest. The SPR and TSR of epoxy resin were higher during the test, while the SPR of epoxy asphalt was higher in the early stage, and then sharply decreased. The TSR was close to that of base asphalt, which was approximately 2400 m2P of base asphalt were the lowest, that is, 0.00635 and 0.1639 g/s, respectively. In contrast, the values of COP and CO2P of resin materials were higher, which was mainly caused by high-temperature fracture and combustion of the polymer chain.The values of COP and COAccording to the results of heat release and smoke release tests, the combustion characteristics of epoxy asphalt were not between those of base asphalt and epoxy resin; instead, they promoted, interacted with, and influenced each other. In addition, although epoxy asphalt was the equal mass synthesis of base asphalt and epoxy resin, the pyrolysis and combustion characteristics of epoxy asphalt were closer to that of epoxy resin, indicating that the influence of epoxy resin was greater than that of base asphalt.To further understand the combustion mechanism of different materials, the residual carbon was examined using the cone calorimeter method and was sprayed with gold, and SEM  was used. The accelerating voltage was 20 kV, amplified 50, 200, and 2000 times to observe the surface morphology. The morphology of residual carbon is shown in At high temperature, the surface of base asphalt will flow and agglomerate, resulting in molten droplets, which accelerates the endothermic decomposition of asphalt. After combustion, the surface of residual carbon presented a uniform high-low fluctuation shape, which is a skeleton pore structure and has many through holes with a large pore diameter and loose structure. This is mainly caused by the precipitation of internal decomposition gas breaking through the barrier layer during asphalt combustion. Notably, more through holes indicate more volatile matter in gas phase. Under a high-power microscope (2000× magnification), it can be observed that the residue had a fine-scale structure, which may be formed by stacking collapsed structures after decomposition.The carbon residue rate of epoxy resin after combustion was low, but the carbon residue surface was dense and flat with few through holes. The main reason for this is that the volatile content was relatively small, and the 2 and CO2 released by polymers after combustion on the carbon layer.It can be seen from (1)Epoxy asphalt exhibited different characteristics in pyrolysis and combustion stages. When it was close to the peak temperature of each stage, a peak value was observed in the ion flow curve, and the release of volatiles was the largest.(2)2, 104.5 MJ/m2, 0.203m2/s, and 2995.2 m2/m2, respectively, and the combustion was intense. However, the smoke produced from epoxy resin reduced to a certain extent after the base asphalt was added in the epoxy resin in the form of an interpenetrating network. The SPR of epoxy asphalt was high in the early stage, up to 0.220 m2/s, then sharply decreased. Moreover, the TSR was close to that of base asphalt.The peak HRR, THR, SPR, and TSR of epoxy resin were high, reaching 1015.7 kW/m(3)2 and CO2 released by polymers after combustion on the carbon layer.The residual carbon rate was low, and the volatile content was relatively small after the combustion of epoxy resin. Its combustion form is to deepen the combustion layer-by-layer at the pore-forming place. Therefore, the residual carbon surface was dense and flat with few through holes. However, the carbon residue surface of epoxy asphalt after combustion exhibited an irregular network structure, which was rough and loose, with some holes; nevertheless, most of them existed in the form of embedded nonpenetration, which is mainly caused by the impact of noncombustible gases such as N(4)The results of heat release and smoke release tests showed that epoxy asphalt is not a simple fusion of base asphalt and epoxy resin. Instead, they promote, interact with, and affect each other, and the influence of epoxy resin on the pyrolysis and combustion characteristics of epoxy asphalt was greater than that of base asphalt.In this study, the pyrolysis and combustion characteristics of epoxy asphalt were examined using TG-MS, cone calorimeter, and SEM. The main conclusions are as follows:


A unilateral ureteral obstruction (UUO) model was induced by ligating the left ureter of male SD rats. A total of 24 rats were randomly divided into the following four groups: sham group, model group, SKI group, and benazepril group. The rats in each group were treated for 28 days, and renal function was evaluated by blood urea nitrogen (BUN) and serum creatinine (Scr). The degree of renal fibrosis was assessed by hematoxylin and eosin (HE) and Masson staining. Extracellular matrix (ECM) deposition was evaluated by immunohistochemistry. Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting were used to detect the expression of genes and proteins in the Wnt/β-catenin signaling pathway. Further studies were performed in vitro using HK-2 cells treated with TGF-β1. At 28 days postoperation, the levels of BUN and Scr expression were significantly increased in the UUO group. SKI and benazepril reduced the levels of BUN and Scr, which displayed protective renal effects. Pathological staining showed that compared with the sham operation group, the renal parenchymal structure was severely damaged, the number of glomeruli was reduced, and a large amount of collagen was deposited in the kidney tissue of the UUO group. SKI treatment reduced morphological changes. Immunohistochemistry showed that compared with the sham operation group, the content of collagen I and FN in the kidney tissue of the UUO group were significantly increased, whereas the SKI content was decreased. In addition, compared with the UUO group, the levels of Wnt1, active β-catenin, Snail1, and PAI-1 expression were reduced in the SKI group, suggesting that SKI may reduce renal fibrosis by mediating the Wnt/β-catenin pathway. Further in vitro studies showed that collagen I, FN, and α-SMA levels in HK-2 cells were significantly increased following stimulation with TGF-β1. SKI could significantly reduce the expression of collagen I, FN, and α-SMA. A scratch test showed that SKI could reduce HK-2 migration. In addition, by stimulating TGF-β1, the levels of Wnt1, active β-catenin, snail1, and PAI-1 were significantly upregulated. SKI treatment could inhibit the activity of the Wnt/β-catenin signaling pathway in HK-2 cells. SKI improves kidney function by inhibiting renal fibrosis. The anti-fibrotic effects may be mediated by regulation of the Wnt/β-catenin pathway and EMT inhibition.Shenkang Injection (SKI) is a traditional Chinese medicine injection commonly used in the clinical treatment of chronic kidney disease. Although it has been confirmed that SKI has anti-kidney fibrosis effects, the underlying mechanism remains unclear. To investigate the effects of SKI on epithelial-mesenchymal transition (EMT) and Wnt/ Renal fibrosis is a common end-stage pathology associated with various chronic kidney diseases (CKD) and is expected to become the fifth leading cause of death in the world by 2040 . Therefoα-SMA synthesis, as well as cellular migration and invasion [Renal fibrosis has complex etiology and pathogenesis, among which the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the most important mechanisms involved in renal fibrosis. Renal epithelial cells are transformed from mesenchymal-to-epithelial transition (MET) during embryonic development , 4. It winvasion . It is binvasion . Therefoβ-catenin pathway is an indispensable, evolutionarily conserved pathway involved in the process of nephron formation. In normal adult kidneys, the Wnt/β-catenin pathway is quiescent and can be reactivated following kidney injury [β-catenin is a multifunctional cytoskeletal protein that exists in several different cell types, including endothelial cells, fibroblasts, and osteoblasts, in which it regulates their proliferation, differentiation, and apoptosis [β-catenin expression is associated with CKD [β-catenin is activated, it serves as an effector molecule to amplify Wnt signaling. In β-catenin dephosphorylation, the free β-catenin was accumulated, and epithelial integrity was destroyed [β-catenin signaling pathway may represent a potential treatment strategy for renal fibrosis.The Wnt/y injury –19. Thisy injury , 21 havepoptosis , 23. Morwith CKD , 24, 25.estroyed . TherefoAstragalus mongholicus Bunge, Rheum officinale Baill, Salvia officinalis L, and Carthamus tinctorius L. Moreover, SKI was developed from “Shenkang formula (SKF),” which was first used by Professor Chuan-hui Ye. SKI has been widely used clinically to treat chronic kidney disease [β/Smad3 signaling pathway in 5/6 nephrectomy [β-catenin signaling pathway. In particular, the effect of SKI on rat renal tissue fibrosis and HK-2 cellular trans-differentiation was observed, for which the clinical application of SKI in anti-kidney fibrosis treatment provides evidence.Shenkang injection (SKI) is composed of four traditional Chinese herbs:  disease . Studies disease –30. In a disease . SKI cou disease . In addi disease . MoreoveA total of 50 eight-week-old male SD rats weighing 210 g ± 20 g, were purchased from Chengdu Dashuo Experimental Animal Co. Ltd. The rats were raised in separate cages at the Animal Experiment Center of Chengdu University of Traditional Chinese Medicine. The temperature was controlled at 20°C–25°C; the humidity was maintained at 50%–60%; and a density of six rats per cage. Rats were free to eat and drink for a week. The study was conducted under the guidance of the Ethics Committee of Chengdu University of Traditional Chinese Medicine. Every effort was made to reduce animal suffering.n = 11 per group). In the sham group, only the left kidney was exposed during the operation. The dose of SKI was administered to rats at a dose of 0.9 mL/day, which was half the equivalent dose for clinical use. Benazepril rats were administered at a clinically therapeutic equivalent dose of 0.9 mg/kg. Benazepril was dissolved in distilled water into a 1 mL suspension for intragastric administration. After treatment for 28 days, 24 rats in the sham group, model group, SKI group, and benazepril group (6 per group) survived and were included in the group for data analysis.Rats underwent a unilateral ureteral obstruction (UUO) operation. The experiment was completed under aseptic conditions, and the specific UUO steps were performed as follows: the rats were placed in the supine position after anesthesia. A longitudinal incision was made 1 cm next to the midline of the abdomen. The left kidney was located; the surrounding fat tissue was peeled off; the ureter was freed; and the upper 2/3 section of the ureter was ligated with a 4-0 surgical silk suture and ligated again approximately 5 mm apart to ensure the success of the model , 35. A tAstragalus, and Salvia. The medical extraction and preparation of SKI obtained Chinese patent certification and protection. In the same batch, the main components were subjected to standardization. Each 20 mL SKI contained 6 g of raw medicine, and the equivalent dosage of SKI was 1.8 mL/day. Each dosage of SKI was prepared in a 0.9 mL/d liquid for the administration of an intraperitoneal injection. In preliminary experiments, three drug concentrations were selected: high (3.6 mL/d), medium (1.8 mL/d), and low (0.9 mL/d). The effect of the high-dose treatment effect was not good, and both the middle- and low-dose treatment effect was equivalent. Finally, the low dose was selected for further research into its therapeutic effect. Both fingerprint and chromatographic analyses were conducted to perform a chemical and quantitative analysis of SKI [SKI was purchased from Xi'an Century Shengkang Pharmaceutical Co. Ltd. The batch number was 202012113 and contained the following four traditional Chinese medicine extracts: Safflower, Rhubarb, s of SKI , 37. BenA veterinary biochemical analyzer was used to detect serum creatinine, 28 days after the operation . BUN was tested using a commercial kit . The rat body weight and kidney weight were also measured with an electronic scale (i2000) and electronic balance (HANGPING JA3003) 28 days postoperatively, respectively.The kidney was cut vertically into two parts along the largest longitudinal section. One half was fixed with 10% formaldehyde, and the renal tissue was processed by hematoxylin-eosin (HE) and Masson staining. The pathological sections were observed under a microscope . HE staining was used to count the number of glomeruli. A total of 10 separate views (100×) of the HE stained sections were selected, and the number of glomeruli was counted. A total of 6 separate views (100×) of the Masson stained sections were selected to evaluate the degree of renal fibrosis. The collagen volume fraction (CVF) was analyzed using ImageJ software.2 incubator. Under the microscope, the cells were adherent. TGF-β1 is a known fibrosis inducer, and ICG-001 is a known Wnt/β-catenin pathway inhibitor; thus, TGF-β1 could be used to induce EMT in HK-2 cells. ICG-001 can be used in rescue experiments to further confirm the role of the Wnt/β-catenin pathway in renal fibrosis. TGF-β1 was obtained from Abcam (ab50036), and ICG-001 was purchased from AbMole (M2008). To investigate the role of SKI in cell EMT, HK-2 cells were treated with TGF-β1 (10 ng/mL) for 24 h at 37°C. HK-2 cells in the exponential growth phase were digested with 0.25% trypsin-EDTA, passaged, and counted, and the HK-2 cells were inoculated into a six-well plate. Next, either SKI or ICG-001 was mixed with TGF-β1. After 24 h of stimulation, the cells were collected for further analysis.The normal human kidney immortalized proximal tubule epithelial cell line, HK-2 cells, were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences  and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a 5% CO3 cells/well. After overnight incubation at 37 C, the samples were divided into a control group and an experimental group. The control group was cultured normally, whereas the experimental group was treated with 10, 5, 2.5, 1.25, and 0.75 mL SKI. Next, CCK-8 reagent and serum-free solution (1 : 10) medium were added to each well, followed by incubation for 1 h at 37°C in 5% CO2. A VARIOSKAN microplate reader (Thermo Electronics Corporation) was used to quantify the optical density (OD) at 450 nm.CCK-8 was used to select the appropriate concentration of SKI drugs. HK-2 cells were seeded into each well of a 96-well plate at a density of 6 × 104 cells/well). At 80%–90% confluency, the cellular monolayer was scraped with a sterile 200 μL suction head and cultured under standard conditions for 24 h. Wound healing was captured at 0 h and 24 h under a microscope, and the scratch ratio was calculated using the following formula: acratch ratio = (area 0 h – area 24 h)/area 0 h.Cell motility was assessed by a wound healing measurement. The cells were cultured in six-well plates  labeled phalloidin  in accordance with the manufacturer's instructions. The labeled cells were analyzed using a Nikon ECLIPSE Ti .α-SMA, and E-cadherin were detected using IHC. The steps are as follows: paraffin sectioning, deparaffinization, antigen recovery, and serum blocking. The primary antibody of the target protein  and HRP donkey anti-mouse IgG  were added after washing. SABC was added and washed again, and DAB was added for color development and dehydration and mounted after counterstaining. Six individual sections were randomly selected to calculate the integrated optical density.Collagen I, FN,  protein  was addeα-SMA , and active β-catenin  antibodies and incubated with primary antibodies at 4°C overnight. The cells were incubated with the corresponding secondary antibody for 1 h at room temperature. Mouse (FITC goat anti-mouse IgG (H + L); AS001, 1:50) or rabbit (FITC goat anti-rabbit IgG (H + L); AS011, 1:50) antibodies were incubated with secondary antibodies. DAPI was used to counterstain the cell nucleus, and an anti-fluorescence quencher was added and was mounted on a glass slide. HK-2 cells were observed under Nikon ECLIPSE Ti .The cultured HK-2 cells were fixed with 4% paraformaldehyde and washed three times with 0.1% Triton X-100. The cells were combined with preconfigured anti-E-cadherin , anti-μL, including 6.4 μL DNase-free (dd) water, 10 μL Mix SYBR (QP-01014 FOREGENE), 0.8 μL forward and reverse primers, and 2 μL cDNA diluent. The absorbance of SYBR green fluorescence was detected in each tube at the end of each cycle. The cycle threshold (CT) values were obtained. The primers used in this study are shown in α-SMA, E-cadherin, Wnt1, PAI-1, β-catenin, and Snail1. The primers were supplied by Tsingke Biotechnology Co. Ltd. The relative expression values of the target genes were calculated using the 2−ΔΔCt method [The total RNA was extracted using an animal total RNA isolation kit . A ScanDrop 100 was used to detect the level of RNA purity. cDNA was synthesized using a Master PremixRT EasyTM II (RE-01022 FORE GENE). qPCR  displayed the following thermal cycle curve: 95°C for 3 min, 95°C for 10 s, and 40 amplification cycles . The reaction system consisted of 20 t method .α-SMA, E-cadherin, Wnt1, PAI-1, β-catenin, active-β-catenin, and Snail1 were the results of the relative internal reference proteins. The primary antibodies used in this study are shown in The renal tissue was homogenized in radioimmunoprecipitation assay (RIPA) protein lysate containing protease and phosphatase inhibitors. After centrifugation , the supernatant was retained, and its total protein content was determined according to the bicinchoninic acid  kit instructions. A 5 × SDS-PAGE loading buffer was used to balance the loading volume of each well to 40 mg/10 mL. Next, 5 × protein loading buffer was added to each sample solution, mixed well, boiled at 100°C for 15 min, cooled, and stored at −20°C until subsequent use. The samples were subjected to 10% SDS-PAGE gel, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked in phosphate-buffered saline Tween 20 (PBST) containing 5% bovine serum albumin (BSA) at room temperature for 1.5 h. Primary antibodies were incubated with the membranes at 4°C for 12 h, after which the membrane was incubated with the secondary antibody at room temperature for 1.5 h. The immunoreactive protein bands were visualized using ECL  chemiluminescence method for color rendering. An E-blot electronic tablet imager was used for membrane visualization and analysis . The final reported data for collagen I, P < 0.05 indicated a statistically significant difference. SPSS 20.0 statistical software  was used for data analysis.Continuous data were expressed as the mean ± standard deviation. A one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and the least significant difference-t test (LSD-t test) was used for pairwise comparisons. A threshold of P < 0.001). The wet weight of the left kidney was significantly increased (P < 0.001). After SKI treatment, the levels of BUN and Scr in the rats were significantly lower than those in the UUO group (P < 0.001), and the wet weight of the left kidney was mildly decreased (P < 0.05). These results indicate that SKI can improve the renal function impaired by UUO surgery.The renal function of rats reacted by BUN and Scr Figures . The levP < 0.001). In addition, the number of glomeruli was increased to 51.33 (P < 0.05) following SKI treatment. Benazepril exhibited similar effects to that of SKI. Masson staining revealed extensive collagen deposition in the UUO group; however, SKI treatment greatly reduced the amount of collagen deposition in UUO rats (P < 0.001). After SKI treatment, CVF was decreased by 58.14% (P < 0.001). The level of collagen I and FN expression in the kidney tissue was evaluated by immunohistochemical methods . After SKI treatment, the IOD of type I collagen and FN decreased to 12% and 19% (P < 0.001). Compared with the UUO group, the level of collagen I protein expression, Col1a1, and Fn1 mRNA expression in the SKI group was also decreased , whereas the IOD of E-cadherin (P < 0.001). Compared with the UUO group, the level of Acta2 RNA and protein expression in the SKI group were also reduced (Cdh1) RNA and protein expression , β-catenin (P < 0.001), active β-catenin (P < 0.01), Snail1 (P < 0.001), PAI-1 (P < 0.01), and TCF4 (P < 0.001) protein expression were significantly increased . SKI treatment could reduce the level of Wnt1 (P < 0.01), β-catenin (P < 0.001), active β-catenin (P < 0.05), Snail1 (P < 0.01), PAI-1 (P < 0.05), and TCF4 (P < 0.01) expression.The Wnt/ Figures . The exp Figures  was signβ1 was further studied. The model group of HK-2 cells was cultured in DMEM with 10 ng/mL TGF-β1, and the treatment group was administered SKI on this basis. RT-qPCR and/or western blot were used to detect the levels of collagen I and FN. The results showed that compared with the cells cultured in DMEM, the cells secreted more Col1a1 and Fn1 following treatment with TGF-β1  is strongly suggestive of EMT. The results showed that , compared with cells treated with DMEM. The scratch ratio was downregulated to 0.33 (P < 0.001) in the treatment with SKI  in the treated with ICG-001 , β-catenin (P < 0.01), active β-catenin (P < 0.001), Snail1 (P < 0.001), PAI-1 (P < 0.01), and TCF4 (P < 0.01) were dramatically increased compared with the cells cultured with DMEM (P < 0.05), β-catenin (P < 0.01), active β-catenin (P < 0.05), Snail1, PAI-1 (P < 0.05), and TCF4 (P < 0.05) mRNA . These results confirmed that SKI could reduce fibrosis by inhibiting the Wnt/β-catenin signaling pathway.After inhibiting the Wnt/d by SKI . RT-qPCR Figures  expressi Figures  expressi Figures . The scr Figures . As showpression . The expith DMEM . SKI cou05) mRNA . The levα-SMA in HK-2 cells, as well as reduce the migration of HK-2 cells treated with TGF-β1 and restore phenotypic changes. Thus, SKI may be related to EMT suppression.Renal fibrosis represented the major hallmark of nearly all progressive chronic kidney diseases. EMT was first discovered to be associated with fibrogenesis in renal tubular epithelial cells of a mouse anti-tubular membrane disease model . The mouβ-catenin pathway is a canonical pathway involved in the progression of fibrosis. In addition, β-catenin is a multifunctional cytoskeletal protein, which forms a cell adhesion link complex with E-cadherin to achieve epithelial integrity. Wnt binds with the receptor in the plasma membrane to induce a destruction complex composed of GSK-3β, CK1, APC, Axin, and β-catenin changes. In addition, the phosphorylation of GSK-3β can activate β-catenin to mediate Wnt canonical signaling. The level of active β-catenin in the cytoplasm was increased and subsequently translocated into the nucleus where it combined with TCF/LEF to induce the transcription of fibrosis-related genes, including Snail1, PAI-1, and FN. Snail1 is a key transcription factor driving EMT, and it cannot only inhibit the expression of E-cadherin but also causes the destruction of epithelial cell-cell adhesion [β-catenin are upregulated in the renal tubular epithelium of human and animal fibrotic kidneys [β-catenin activation induces Snail1 expression in both renal tubular epithelium cells and podocytes. PAI-1 participates in inflammation and plays a vital role in the pathogenesis of fibrogenesis, and FN is a component of ECM. In addition, other genes  also play a vital role in fibrogenesis. SKI can improve renal function and inhibit β-catenin, Snail1, PAI-1, TCF4, and Wnt1 expression. Our results show Wnt/β-catenin signaling pathway activation in HK-2 cells following stimulation with TGF-β1, which resulted in the production of active β-catenin. Active β-catenin promotes Snail1, PAI-1, and TCF4 expression. In addition, SKI can inhibit TGF-β1-induced activation of the Wnt/β-catenin signaling pathway in HK-2 cells and reduce β-catenin activity. Therefore, we speculate that SKI may play a role in inhibiting the activation of β-catenin.The Wnt/adhesion . Studies kidneys , 40. In β-catenin signaling pathway. β-catenin may represent an attractive treatment target for renal fibrosis, and improving EMT may have favorable anti-fibrosis effects. This research provides basic mechanistic support for the clinical application of SKI.In summary, our in vivo and in vitro research showed that SKI could improve renal function and exhibit anti-fibrotic effects. The underlying mechanism might be related to inhibiting the Wnt/


Hair loss, or alopecia, is a dermatological disorder that causes psychological stress and poor quality of life. Drug-based therapeutics such as finasteride and minoxidil have been clinically used to treat hair loss, but they have limitations due to their several side effects in patients. To solve this problem, there has been meaningful progress in elucidating the molecular mechanisms of hair growth and finding novel targets to develop therapeutics to treat it. Among various signaling pathways, Wnt/β-catenin plays an essential role in hair follicle development, the hair cycle, and regeneration. Thus, much research has demonstrated that various natural products worldwide promote hair growth by stimulating Wnt/β-catenin signaling. This review discusses the functional role of the Wnt/β-catenin pathway and its related signaling molecules. We also review the molecular mechanism of the natural products or compounds that activate Wnt/β-catenin signaling and provide insights into developing therapeutics or cosmeceuticals that treat hair loss. Hair loss is a common medical problem that occurs due to both hair density loss and the thinning of hair. This phenomenon is caused by hormonal and non-hormonal problems ,2. Hair Currently, both minoxidil and finasteride, which are approved by the Food and Drug Administration, contribute to promoting hair regrowth or preventing hair loss. In total, 2–3% or 5% topical minoxidil has been used to treat AGA in females and males, respectively . As finaThe hair follicle is a complex structure and undergoes a cycle of hair growth , regression , and rest  under the regulation of various signaling pathways: the wingless-type MMTV integration site family member Wnt/β-catenin, transforming growth factor (TGF)-β, fibroblast growth factor (FGF), sonic hedgehog (SHH), bone morphogenetic protein (BMP), and Notch signaling. These various signaling pathways have been implicated in hair regeneration ,9. In paBased on these molecular targets, novel, active ingredients are required to retard or prevent hair loss. Thus, various studies addressing the increasing problem of hair loss using natural products with few side effects have been examined. Many researchers have identified natural compounds such as polyphenols and natural plants found worldwide as potential active ingredients for treating hair loss ,13,14,15Generally, a human scalp maintains hair by regulating the hair cycle . A hair Hair development involves crosstalk between the epidermal and dermal compartments in the hair follicle ,25. The In the canonical Wnt/β-catenin signaling pathway, the Wnt proteins are associated with the Frizzled receptor and a low-density lipoprotein-related protein (LRP) or receptor tyrosine kinase. This complex inactivates glycogen synthase kinase-3β (GSK-3β) and stabilizes β-catenin to avoid ubiquitin-dependent degradation ,28. The Dickkopf 1 (DKK1), a Wnt antagonist, inhibits Wnt action by binding to the LRP ,31. DKK1A previous study examined the effects of bone marrow mesenchymal stem cells (BM-MSCs) overexpressing Wnt1a on the regeneration of depilated mouse hair follicles . The tre−/− mice exhibited metabolic waste syndrome and various skin lesions, such as a disrupted epidermal barrier and alopecia. Stoffel et al. found that the absence of palmitoleic acid (9Z-16:1) in the lipid analysis of Scd1−/− mice blocked the posttranslational O-palmitoleoylation of Wnt3a protein and was necessary for stem cell lineage in the developmental progress of the epidermal barrier and hair growth cycle. In the experiment, an artificial lipid barrier was used as a substitute for the disintegrated epidermal lipid barrier, and remarkably, it activated hair bulge progenitor cells and recovered a regular hair growth cycle in Scd1−/− mice [Kishimoto et al. demonstrated that the overexpression of Wnt3a stimulated β-catenin activity and enhanced hair growth in nude mice with transplanted skin containing DP and keratinocytes . Stearoy−/− mice .+ T cells were enhanced in alopecia areata patients during the transition from the mid-anagen stage to the late catagen stage. These results suggest that both epithelial and mesenchymal interactions in the hair follicle may regulate the immune response of the hair follicle [A previous study demonstrated that macrophage-extracellular vesicles (MAC-EVs), including Wnt3a and Wnt7b, enhanced the proliferation rate of DPCs, and elevated the expression levels of Axin2 and Lef1 . Interesfollicle .During the anagen phase, the gene expression levels of Wnt ligands—including WNT3, WNT4, and WNT10B—and Wnt target genes—including Axin2 and Lef1—significantly increase in DPCs and secondary hair germ cells . On the Wnt5a belongs to a non-canonical Wnt family and generally antagonizes the function activity of canonical Wnt signaling in other tissues. In hair follicles, Wnt5a is significantly expressed in the bulge region and secondary hair germ cells in the telogen phase . A previWnt10b stimulated the differentiation process of skin epithelial cells into the hair shaft by stimulating β-catenin activity . InteresIn AGA, androgens impair the differentiation of HFSC by suppressing Wnt signaling . In bothPrecise intercellular signaling between epithelial cells and mesenchymal cells is essential for the formation of hair follicles. Several intra- and intercellular signaling molecules crosstalk with the Wnt/β-catenin signaling pathway and play a key role in regulating hair follicles ,57,58,59BMP signaling suppresses the onset of anagen ,54. A prThe administration of epidermal growth factor (EGF) enhanced the β-catenin level in the nucleus and stimulated Wnt10b, β-catenin, and EGF receptor. EGF also elevated the expression levels of survivin, Msx2, and SGK3, which were follicle-regulatory genes. The inhibition of β-catenin activity caused by XAV-939 blocks EGF-induced proliferation in ORS cells . A previSimilarly, a previous study revealed that the topical administration of FGF improved hair growth by inducing the initiation of anagen and prolonging the anagen phase in C57BL/6 mice . FGF treHedgehog (HH) and TGF-β, which modulate epithelial-mesenchymal communication in hair follicles, play roles in the regeneration of hair follicles and hair cycling ,58,59. TThe connective tissue growth factor (CCN2) acts as a physiological inhibitor in hair follicle formation and maintains the quiescence status of stem cells. The CCN2 is exclusively expressed in both ORS cells and DPCs. The ablation of CCN2 shortens the telogen phase and enhances the number of hair follicles. On the contrary, the recombinant CCN2 destabilized the β-catenin signaling pathway and diminished the proliferation rate of keratinocytes . Many studies have demonstrated that natural plants can stimulate the Wnt/β-catenin pathway or its related biomarkers to treat hair loss . Aconitie Ciliare Tuber (ACT) extract was found to promote ALP activity and the proliferation rate of immortalized DPCs [Centipeda minima (L.) A. Braun and Asch are traditional Chinese medicines [Centipeda minima (CMX) stimulated the proliferation of human follicle DPCs and increased the expression levels of Wnt5a, Frizzled receptor, and vascular endothelial growth factor (VEGF) [zed DPCs . ACT extzed DPCs . Centipeedicines . A recenr (VEGF) . Furtherr (VEGF) . Ginkgolr (VEGF) . Anotherr (VEGF) .Malva verticillata (M. verticillata) enhanced Wnt reporter activity and elevated β-catenin levels in human DPCs [M. verticillata, promoted the proliferation rate of DPCs and elevated the transcription levels of HGF, keratinocyte growth factor (KGF), insulin-like growth factor-1 (IGF-1), and VEGF. In addition, myristoleic acid upregulated the phosphorylation levels of both Akt and p38 [M. verticillata seed extracts. They identified oleic acid and linoleic acid in the M. verticillate (MH)2. Treatment with linoleic acid stimulated Wnt/β-catenin signaling and promoted human follicle DPC growth [Polynesian cosmetopoeia, Fagraea berteroana, Calophyllum inophyllum, and Bidens pilosa, either upregulate Lef-1 and PPARD genes and/or downregulate DKK1 and TGFB1 genes [Seed extract from man DPCs . Myristo and p38 . This grC growth . LinoleiB1 genes . Three nB1 genes . Prunus mira Koehne (P. mira) seed oil belongs to the Rosaceae family [P. mira nut oil promotes hair follicles into the anagen phase, upregulating the expression levels of Wnt10b and β-catenin. Topical treatments with P. mira nut oil significantly enhanced dermal thickness, hair length, and hair weight compared with minoxidil in the depilated dorsal skin of C57BL/6 mice. They demonstrated that P. mira could promote hair growth in mice by activating the Wnt/β-catenin pathway [Polygonum multiflorum (PM), one of the flowering plants in the buckwheat family, Polygonaceae, dose-dependently elevated the cell viability and mitochondrial activity in human DPCs. PM extract significantly increased the expression level of Bcl2 in DPCs and DPC spheroids. PM extract increased VEGF expression but decreased Dkk-1 expression [e family ,69. P. m pathway ,69. The pression . A red gpression , which epression .Salvia plebeia R. Brown (Labiatae) has diverse biological activities [Salvia plebeia (SP) significantly elevated the proliferation rate of human DPCs compared with the control group. SP increased the expression level of HGF, whereas it decreased the expression levels of TGF-β1 and SMAD2/3. SP stimulated Wnt/β-catenin signaling by increasing the nuclear transfer of β-catenin. SP induced the anagen phase, which resulted in increased hair growth in male C57BL/6 mice [Allium ascalonicum L.) is a traditional medicine for improving hair growth in Thai folkloric wisdom [SRD5A1 and SRD5A2. Conversely, it upregulated the expression level of β-catenin, VEGF, and sonic hedgehogs such as SHH, SMO, and GIL1 and promoted hair growth activity [Thuja orientalis (TO) has been used to treat hair loss patients in East Asia. In one study, TO extract stimulated the anagen phase and improved hair growth in C57BL/6 N mice. The TO-extract-treated group increased β-catenin levels and Shh proteins compared with the control group [Brassicaceae [A previous study found that tivities . Salvia L/6 mice . Shallotc wisdom . Shallotactivity . Thuja ool group . Watercrsicaceae . A recensicaceae . WCE sigsicaceae .Many studies have found many natural compounds that activate the Wnt/β-catenin pathway or its crosstalk, which may be potential therapies to treat hair loss . Fabaceae and Zingiberaceae herbs [+, Lef1+, and Gli1+ [Caesalpinia sappan L. (Leguminosae), is known to possess anti-allergic, anti-inflammatory, and antioxidant properties [+ cells near hair follicles [2O2-induced damage of hDPCs [Houttuynia cordata, possesses hair growth activity in human DPCs [O-caffeoylquinic acid (TCQA), a polyphenolic compound, promoted the hair growth cycle in human DPCs and mouse models [Silybum marianum, has anti-inflammatory and antioxidant properties that can improve various skin disorders [2O2-induced cellular damage in human DPCs [In particular, many polyphenol compounds, including flavonoid and chalcone, contributed to countering hair loss by stimulating the Wnt/β-catenin signaling pathway ,88,89,90ae herbs . The topnd Gli1+ . Baicalind Gli1+ . Anothernd Gli1+ . Additiond Gli1+ . They dend Gli1+ . 3-Deoxyoperties . A previoperties . The 3-Doperties . Epigalloperties ,82. EGCGoperties . EGCG inoperties . Kubo etoperties . These poperties . In a suollicles . Fisetinollicles . Anotherman DPCs . In one man DPCs . A previe models . The TCQe models . SilibinSeveral terpenoid compounds have promoted hair growth by stimulating the Wnt/β-catenin signaling pathway ,92,93. LMagnolia, is known to have anti-inflammatory and anti-angiogenic activities [552 and Ser675) and GSK3β (Ser9). MA enhanced the phosphorylation level of ERK. MA-induced ERK phosphorylation led to alterations in DPC proliferation [Cornus officinalis, has various biological properties [In addition, many studies have demonstrated that various natural products effectively activate the Wnt/β-catenin signaling pathway against hair loss ,103,104.tivities . A previtivities . 5-bromotivities . BDB eletivities . Sinapictivities . Sinapictivities . MA alsoferation . MorroniThere were a few clinical studies of these natural products, such as Watercress , EGCG 8, and valUntil 30 October 2022, PubMed searches were conducted for articles that studied the effects of natural compounds on hair growth. To reflect the latest studies, the time frame of the search was limited from the year 2000 to the present (within 22 years). The search combined the keywords “hair growth”, “Wnt“, “alopecia”, “plant”, “polyphenol”, “flavonoid”, “natural compound”, “dermal papilla cells”, and “hair follicle”. Inclusion criteria are as follows: (1) published in English; (2) intervention included natural compounds or plants; (3) hair growth or alopecia; (4) hair follicle or dermal papilla cells.Data about natural products were chosen from selected references  as folloHair loss, or alopecia, is a common disorder observed among people worldwide. The side effects of finasteride and minoxidil have led to many studies elucidating the molecular mechanisms of hair development, the hair cycle, and growth. Among multiple molecular mechanisms, the Wnt/β-catenin signaling pathway is well-known as an essential pathway in hair regeneration. Accordingly, many studies have identified natural products or compounds worldwide as potential active Wnt/β-catenin candidates for treating hair loss. If these natural products reduce the side effects of preventing hair loss, they will be more effective than minoxidil and finasteride. However, since hair regeneration is performed through a precise network between the epidermal and dermal compartments, the combination of Wnt/β-catenin activators and other candidate drugs, such as BMP inhibitors and TGF-β inhibitors, may help treat hair loss. Thus, future studies must be clinically applied without affecting efficacy or causing the side effects of these natural products. These studies will contribute to developing therapeutics or cosmeceuticals for improving hair growth.


Furthermore, 1644 DEGs were identified between steatotic cells and Ex-4-treated cells, with liver X receptor/retinoid X receptor (LXR/RXR) (p = 2.02 × 10−7) and FXR/RXR (p = 3.28 × 10−7) activation being the two top canonical pathways. The top molecular and cellular functions between untreated and steatotic cells were lipid metabolism, molecular transport, and small molecular biochemistry, while organismal injury and abnormalities, endocrine system disorders, and gastrointestinal disease were the top three molecular and cellular functions between Ex-4-treated and steatotic cells. Genes overlapping steatotic cells and Ex-4-treated cells were associated with several lipid metabolism processes. Unique transcriptomic differences exist between steatotic cells and Ex-4-treated steatotic cells, providing an important resource for understanding the mechanisms that underpin the protective effect of GLP-1RAs on NAFLD and for the identification of novel therapeutic targets for NAFLD.No therapy exists for non-alcoholic fatty liver disease (NAFLD). However, glucagon-like peptide receptor agonists (GLP-1RAs) showed a beneficial effect on NAFLD, although the underpinning mechanisms remain unclear due to their pleiotropic effects. We examined the implicated signaling pathways using comparative transcriptomics in a cell model of steatosis to overcome pleiotropy. We treated steatotic HepG2 cells with the GLP-1RA Exendin-4 (Ex-4). We compared the transcriptome profiles of untreated steatotic, and Ex-4-treated steatotic cells, and used Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and associated genes involved in the protective effect of Ex-4. Ex-4 treatment significantly reduces steatosis. RNA-seq analysis revealed 209 differentially expressed genes (DEGs) between steatotic and untreated cells, with farnesoid X receptor/retinoid X receptor (FXR/RXR) ( Non-alcoholic fatty liver disease (NAFLD), a disease marked by an abnormal accumulation of triglycerides in ≥5% of hepatocytes independently of alcohol consumption and other competing liver disorders , has becThere is presently no approved pharmacotherapy for NAFLD . Diet anRecently, NAFLD patients treated with glucagon-like peptide-1 receptor agonists (GLP-1RAs) demonstrated a favorable effect on liver fat content ,21,22, iGLP-1 is a multifunctional hormone produced by the L-cells of the small intestine . Due to Despite the reported beneficial effect of GLP-1RAs in NAFLD , the undWe recently reported that the treatment of steatotic human hepatoma HepG2 cells with the GLP-1R agonist Exendin-4 (Ex-4) significantly reduces lipid accumulation and that this effect involves a set of novel long non-coding RNAs . In the The oleic acid (OA) was prepared as in  and no dWe induced steatosis in HepG2 cells, as we recently reported ,37. BrieThe following day, the cells were washed and then incubated for three hours in a fresh DMEM solution containing 400 µM OA supplemented or not with 200 nM Ex-4 . At the end of the experiment, we had three treatment conditions: (1) untreated cells (UCs); (2) steatotic cells (StCs), i.e., cells treated with OA for a total of 19 h; and (3) Ex-4-treated steatotic cells (Ex-4TStCs), i.e., cells treated with OA for 19 h and the DMEM was supplemented with Ex-4 during the last 3 h. All conditions were prepared in triplicate. We used the Pure Link RNA Mini kit  to extract the total RNA from UCs, StCs, and Ex-4TStCs, with conditions according to the manufacturer’s instructions. The RNA samples were immediately frozen at −80 °C until use. Before library preparation, we used an RNA broad range assay kit  and Qubit 2.0  to measure the RNA concentration. We assessed the RNA quality with an Agilent RNA 6000 Nano Kit  and Agilent 2100 Bioanalyzer  as per the manufacturer’s instructions. The method to prepare the library for RNA sequencing is reported in . Brieflyp-value ≤ 0.05.We used CLC Genomics Workbench Software Version 21.0.4.  to look for differences in gene expression between untreated, steatotic, and Ex-4 treated steatotic cells. After retrieving raw RNA sequencing data, pair-end reads were aligned to the Hg38 human reference genome. TPM (Transcript Per Kilobase Million) mapped reads were used to calculate the amount of transcript expression. The ANOVA test was used among the three groups to identify differentially expressed genes (DEGs) with a 2-fold change in absolute value. A statistically significant difference was considered as a p-values < 0.05 (Benjamini-Hochberg method) were considered significant. The Venn diagrams were created using an online tool, Venny Online tool. Fold changes in RNA expression of ±2 were applied for filtering, and then the DEGs list was subjected to Ingenuity Pathway Analysis (IPA)  to identify specific networks and pathways. Biological functions and signaling pathways with p-value ≤ 0.05. Thus, a total of 209 and 1644 DEGs were identified when comparing StCs to UCs and Ex-4TStCs to StCs, respectively. The Log2 fold change hierarchical clustered heatmap in In total, 15,783, 15,452, and 15,723 expressed genes were detected in the UCs, StCs, and EX-4-TStCs, respectively. Of these genes, 13,780 genes were expressed in all three groups; 304, 1046, and 567 genes were identified commonly between each pair of groups , while 1133, 323, and 331 genes were discovered exclusively for UCs, StCs, and Ex-4TStCs, respectively A. To idep-value) > 1.3). On the other hand, using the 51 upregulated genes, IPA identified cAMP-mediated signaling, apelin liver signaling, wound healing signaling, GP6 signaling, and atherosclerosis signaling pathways as the top five significantly upregulated canonical pathways  > 1.3). The top two up- and downregulated canonical pathways and the molecules involved in each pathway are shown in the table in When we compared the datasets from StCs and UCs, we detected a total of 209 DEGs, with 51 being upregulated and 158 being downregulated . The topFurthermore, IPA identified several molecular and cellular functions when using the 51 upregulated DEGs . Figure A comparison of Ex-4TStCs and StCs datasets identified 1644 DEGs, with 479 being upregulated and 1165 being downregulated . Tables Moreover, IPA identified several molecular and cellular functions when using the 479 upregulated DEGs . Figure To better understand which genes might be functionally related to the protective effects of Ex-4 on OA-induced steatosis, we looked at the list of DEGs that overlap between StCs and EX-4TStCs datasets. As shown in the Venn diagram depicted in Afterwards, we examined the pathway enrichment analysis using the 31 overlapping genes and found that the top five canonical pathways that are significantly enriched include GP6 signaling, hepatic fibrosis/hepatic stellate cell activation, cAMP-mediated signaling, apelin liver signaling, and wound healing signaling A. AdditiBecause of the world’s rising obesity rates, NAFLD has emerged as a major global healthcare concern. There is currently no approved treatment for NAFLD. However, according to recent research, GLP-1R agonists (GLP-1RAs) may help with NAFLD ,39. ThatIn the present study, we employed comparative transcriptomics to identify the putative signaling pathways and molecules implicated in the protective effect of Ex-4 on steatosis in HepG2 cells. Our rationale for using an in vitro steatosis model is twofold: firstly, as a precursor of NAFLD, hepatic steatosis plays a vital role in the pathological process of NAFLD; and secondly, to overcome the weight-inducing effect of GLP-1RAs in vivo and to investigate the impact of direct stimulation of the hepatic GLP-1R, whose expression by HepG2 cells has already been established . IdentifTo the best of our knowledge, this is the first work to look at the signaling pathways regulated in a hepatic cell by directly activating the GLP-1R using a GLP-1R agonist. Our analysis identified key differentially expressed mRNA transcripts and pathways across three cell conditions: (1) untreated (UCs), (2) steatotic (StCs), and (3) steatotic cells treated with Ex-4 (Ex-4TStCs). These findings may help shed important mechanistic light on the protective impact of GLP-1R agonists on NAFLD.We detected several significant DEGs between UCs and StCs and between StCs and Ex-4TStcS. These DEGs are involved in various critical biological functions and signaling pathways germane to lipid metabolism, the primary deranged process in NAFLD. Indeed, NAFLD arises when the uptake of fatty acids (FA) and triglycerides (TG) from circulation and de novo lipogenesis saturate the rate of FA β-oxidation and very-low-density lipoprotein (VLDL)-TG export . Among tThe FXR is a major member of the ligand-activated nuclear receptor superfamily . It is aAs mentioned above, liver de novo lipogenesis is a significant contributor to NAFLD development. FXR-deficient mice exhibited a significant induction of lipogenic genes, such as fatty acid synthase (FAS), SREBP-1c, and stearoyl-CoA desaturase 1 (SCD1) . ConsequIn mammalian cells, fatty acid oxidation (FAO), mediated by β-oxidation in mitochondria and peroxisomes and ω-oxidation in cytochromes, plays a significant role in energy generation, especially in periods of low circulating glucose concentrations . ActivatAnother prominent canonical pathway downregulated in steatotic cells and upregulated after Ex-4 treatment is the LXR/RXR activation pathway. LXRs are cholesterol sensors that play an essential role in inflammatory control as well as in the regulation of fatty acid, cholesterol, and glucose metabolism . LXRα waNevertheless, LXRs exert anti-inflammatory functions in various cells and tissues. For example, Wouters et al.  demonstrWe have found 19 genes that were downregulated in StCs compared to UCs but upregulated after Ex-4 treatment. These 19 genes were enriched in several sub-pathways of lipid metabolism, including lipid droplets, cholesterol homeostasis, lipid catabolic process, cholesterol metabolic process, cholesterol biosynthetic process, positive regulation of cholesterol efflux, cellular response to cholesterol, reverse cholesterol transport, negative regulation of cholesterol storage, and medium-chain fatty acid metabolic process. This observation suggests that the modulation of the expression of these genes by Ex-4 might be crucial for the observed beneficial effect of this drug on steatosis. In the absence of appropriate therapy, NAFLD remains a serious medical condition. GLP-1R agonists have showed potential as NAFLD therapy, although the underlying mechanisms remain unknown. We employed transcriptomics and functional pathway analysis to show that the Ex-4-induced steatosis improvement in HepG2 cells might be partially explained by activation of the FXR/RXR activation pathway via direct stimulation of the GLP-1R. Furthermore, while activation of the LXR/RXR activation pathway by Ex-4 in our model does not reconcile with steatosis reduction in in vivo studies, GLP-1R agonists may employ this pathway to reduce hepatic inflammation and so alleviate NAFLD. Our findings may pave the way for a better understanding of the molecular mechanisms behind the beneficial effect of GLP-1R agonists in NAFLD patients. The main limitation of the present study is the use of the HepG2 cell line instead of primary hepatocytes. Validation of the present results through a comprehensive in vivo investigation of the differentially expressed mRNAs and pathways is warranted in the future.


Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining and cardiac enzyme levels. Hearts were subjected to 30 min regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery followed by 30 min reperfusion. Hearts were treated during reperfusion with either the non-lipidated precursor of tirzepatide (NLT), GLP-1, GLP-1 (9-36), or Ex-4 in the presence or absence of Ex-4 (9-39). Infusion of GLP-1 (9-36) or Ex-4 protected the heart against I/R injury (p > 0.01) by normalizing cardiac hemodynamic and enzyme levels. Neither GLP-1, NLT, nor Ex-4 (9-39) showed any protection. Interestingly, Ex-4 (9-39) blocked Ex-4-mediated protection but not that of GLP-1 (9-36). These data suggest that Ex-4-mediated protection is GLP-1-receptor-dependent but GLP-1 (9-36)-mediated protection is not.Tirzepatide is a dual glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist and a promising therapy for type 2 diabetes mellitus (T2DM). GLP-1 is an incretin hormone with therapeutic potential beyond type 2 diabetes mellitus. However, GLP-1 is rapidly degraded by dipeptdyl peptidase-IV (DPP-IV) to GLP-1 (9-36). Exendin-4 (Ex-4) is a DPP-IV-resistant GLP-1 receptor agonist which, when truncated to Ex-4 (9-39), acts as a GLP-1 receptor antagonist. In the present study, hearts isolated from Wistar rats ( Cardiovascular diseases (CVDs) are the most common cause of death worldwide . IschemiHeloderma suspectum, acts as potent GLP-1R agonist. An amino acid substitution at position 2  in the reptilian peptide results in DPP-IV-resistance and a longer circulating half-life. Removal of the first eight amino acids of Ex-4 results in Ex-4 (9-39), a GLP-1R antagonist that has been used extensively in the study of the physiology of GLP-1 [Glucagon-like peptide-1 (GLP-1) is an incretin hormone released from the gastrointestinal tract in response to feeding and functions primarily to potentiate insulin secretion from pancreatic β-cells in a glucose-dependent manner . As a reof GLP-1 .GLP-1R is widely expressed and GLP-1 is now understood to be a pleiotropic hormone with therapeutic potential beyond type 2 diabetes mellitus. GLP-1R agonists have been shown to be both neuro- and cardio-protective . InteresThe end point for the evaluation of the effect of I/R and its treatments on the heart varies between laboratories. Some research groups use LV hemodynamics and contractility as an end point to evaluate the effect of I/R and its treatment on the heart muscle and its performance. Other groups use the infarct size, which is crucial for the prognosis of the disease, as an end point. Coronary and coronary vascular resistance were also used to evaluate the perfusion of the flow. In this study, for a full picture of the effect, we used LV hemodynamics, contractility, and coronary dynamics as end points. Furthermore, we used infarct size and cardiac enzyme levels as a confirmation of our findings. Heart hemodynamics during stabilization, ischemic, and reperfusion periods were evaluated. Left ventricular dynamics were determined throughout the experiment by measuring LVEDP, maximum DPmax, and LV ±dP/dt. Coronary vascular dynamics were evaluated by measuring CF and CVR. Body weights and LV weights were not significantly different between the experimental groups. The values for cardiovascular dynamics in all study groups were presented as percentages of the baseline data.Left ventricular dynamics, contractility, and coronary vascular dynamics were not significantly different between the groups when at baseline heart perfusion. Ischemia, which followed the baseline perfusion, resulted in a considerable deterioration in the heart functions which was reversed by GLP-1 (9-36) and Ex-4 but not by GLP-1 .The same effect was observed in the LV ±dP/dt, which was significantly improved by the infusion of GLP-1 (9-36) and Ex-4 . These rWe then used NLT, the non-lipidated precursor of tirzepatide, to investigate any cross-talk between the GLP-1 and GIP receptors or possible synergy in their protection. Surprisingly, the infusion of NLT before ischemia or reperfusion did not protect the heart against I/R A–D. ThesTo investigate whether the improvements in the hemodynamics, infarct size, and cardiac enzyme levels reported above were modulated via a signaling pathway involving GLP-1R, the GLP-1R antagonist Ex-4 (9-39) was used at reperfusion in the presence of GLP-1 (9-36) and Ex-4. Ex-4 (9-39) completely blocked the protection given by Ex-4 but not the protection produced by GLP-1 (9-36) . p < 0.05)  and Ex-4 resulted in a significant decrease in the protein levels of TNF-α, IL-1β, and IL-6 compared to those of untreated controls ( < 0.05) . This ef < 0.05) A–C.p < 0.01) increased SOD and CAT levels compared to controls. This was not seen when the heart was treated with NLT and was completely abrogated by Ex-4 (9-39) in the presence of Ex-4 but not when co-administered with GLP-1 (9-36)  and Ex-4 significantly (1 (9-36) A,B. There are currently several GLP-1 receptor agonists used clinically for the treatment of both type 2 diabetes mellitus and obesity, for example, liraglutide, lixsenatide, semaglutide, dulaglutide and exenatide . This clThe present study also demonstrates the protective effects of GLP-1 (9-36) and Ex-4 on the heart against I/R injury. This involved the GLP-1R for Ex-4 but not for GLP-1 (9-36). Both Ex-4 and GLP-1 (9-36) have previously been shown to have cardio-protective effects ,26,27. HNo protection of the heart against I/R injury by native GLP-1 could be demonstrated in the present study. This contrasts with previous studies which report GLP-1-mediated protection of the heart against I/R injury in both animal models of diabetes and diabetic patients ,29. The Intriguingly, the present study demonstrates that the GLP-1 degradation product GLP-1 9-36) displays GLP-1R-independent protection of the heart against I/R injury. As mentioned previously, GLP-1 (9-36) has been reported to be both an antagonist and a weak partial agonist of the GLP-1R as well as being biologically inactive ,31,32. F displaysTreatment of isolated hearts with either Ex-4 or GLP-1 (9-36) at reperfusion protected the heart against I/R injury and normalized LV dynamics and coronary vascular dynamics to those of controls. Whereas the cardio-protective effects of Ex-4 were inhibited by infusion of the GLP-1R antagonist Ex-4 (9-39), this was not the case for GLP-1 (9-36) . These dIn addition, we used NLT in this study to investigate potential improvement in the protective effects compared to Ex-4 and GLP-1 (9-36). Interestingly, infusion with NLT before ischemia or at reperfusion did not protect the heart against I/R injury. NLT was proven to be effective as a therapy for type 2 diabetes, but its effect on the protection of the heart against I/R injury has not yet been reported . AlthougThis study reported an anti-inflammatory effect for Ex-4 and GLP-1 (9-36), as treatment of the heart with these compounds resulted in a significant reduction in TNF-α, IL-1β, and IL-6. Although the presence of immune cells in the isolated heart was unlikely, several studies have shown that I/R causes its damage and cardiac dysfunction to the heart partly by promoting pro-inflammatory cytokine formation ,42. TreaIn this study, we reported an increase in the levels of the antioxidant enzymes SOD and CAT after the treatment with Ex-4 and GLP-1 (9-36), which proves the presence of antioxidant activity in these compounds. Although ROS are essential for many beneficial physiological processes, their contribution to pathological conditions is indispensable , and GLPn = 8 per group) were used in this study. Animal treatment and handling were carried out according to the guidelines of Animal Resource Center of Kuwait University, Kuwait. Rats were maintained at 22 °C on 12 h light/dark cycle (7 am–7 pm), and water and food were unrestrictedly available. Rats were anesthetized with intraperitoneal injections of sodium pentobarbital (60 mg/kg), and heparin (1000 U/kg) was administered through the femoral vein. Heart cannulation and perfusion were performed as described previously [2 (5%) and O2 (95%). Heart instrumentation included placing pacing electrodes on the right atrium (RA) appendage to maintain physiological heartbeat. Regional ischemia was induced by occluding left anterior descending (LAD) coronary artery for 30 min. The LAD was encircled by a snare at 0.5 cm below the atrioventricular groove, and a small, rigid plastic tube was positioned between the heart and the snare to ensure complete occlusion of the coronary artery. Subsequently, the heart was reperfused for 30 min. The flow of the perfusate was measured downstream of the aortic cannula using a “Statham pressure transducer” (P23 Db). Preload was held constant at 6 mmHg under basal controlled conditions, and the perfusion pressure (PP) was held constant at 50 mmHg throughout the experimental procedure in all protocols. Constant PP was ensured electronically by means of the perfusion assembly ), an effective system for the accurate adjustment of PP between 5 mmHg and 150 mmHg with ±1 mmHg accuracy level.Age-matched male Wistar rats weighing between 270 and 300 g ( heparin 00 U/kg weviously . Brieflyn = 8 per group) and subjected to four different experimental protocols. The control group was subjected to ischemia and reperfusion without any other treatment; the second group was treated with NLT (0.1 µM) started 15 min before and continued for 15 min; the third to seventh groups were subjected to control procedures with infusion of (0.1 µM each) Ex-4, NLT, GLP-1 (9-39), GLP-1 (9-36), and Ex-4 (9-39) 5 min before the start of reperfusion and continued for 10 min during reperfusion; and the eighth and ninth groups were treated with GLP-1 (9-36) or Ex-4 in presence of the GLP-1R antagonist Ex-4 (9-39) (0.1 µM), as summarized in A total of 72 hearts were subdivided into 9 groups , LV developed pressure (DPmax), and LV contractility (±dP/dt). The coronary-vascular dynamics were evaluated by the assessment of coronary vascular resistance (CVR) and coronary flow (CF). Cardiovascular functions were measured as described in the previous studies ,59. BrieCoronary flow was continuously measured using an electromagnetic flow probe interfaced with a personal computer and attached to the inflow of the aortic cannula. The continuous monitoring of the coronary flow in ml/min was performed digitally using suitable software and was verified manually by timed collection of coronary effluent. The CVR and hemodynamics data were determined every 10 s using an “online data acquisition program” . By the end of each experiment, hearts were snap-frozen in liquid nitrogen, then stored in −80 °C for further analysis. Coronary effluent was collected manually at the apex of the heart from the coronary outflow in small tubes at the end of the reperfusion phase. The hearts were also collected at the end of reperfusion. All heart samples and coronary effluents were frozen in liquid nitrogen and stored at −80 °C for further analysis.Hearts were collected after 30 min of reperfusion and stored overnight at −20 °C. The next day, the hearts were sliced into 4–5 pieces from apex to base. The slices were then incubated in 1% 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) solution in isotonic (pH 7.4) phosphate buffer and then fixed in 4% formaldehyde. The red and pale non-stained areas of every slice were indicated manually on the image. The infarct size as a percentage of the LV was calculated for every heart. Quantification of the LV and infarct size was performed using ImageJ . Cardiomyocyte injury was confirmed by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) release in the coronary effluent during the reperfusion period, as described previously by Ferrera et al. . g for 15 min at 4 °C. The supernatant containing the proteins was then collected, aliquoted, and stored for further analysis. Protein levels were assayed using bicinchoninic acid protein assay . At the end of the experiments, the hearts were snap-frozen in liquid nitrogen and subsequently stored at −80 °C for further analysis. Left ventricle tissue was homogenized in a lysis buffer  containing protease inhibitor cocktail tablet  and centrifuged at 12,000× n = 4) by UV spectrophotometer activity with RANDOX kits  according to the manufacturer’s instructions. Superoxide dismutase  and Catalase  activity were measured in cardiomyocyte lysate (n = 4) was used to evaluate the expression of the proinflammatory cytokines TNF-α (cat# MBS175904), IL-1 (cat# MBS825017), and IL-6 (cat# MBS355410) using enzyme-linked immunosorbent assay (ELISA). The protein content was measured in the samples, which were diluted as suggested by the manufacturer’s assay protocols for the respective commercial kits .The LV homogenate  was used for repeated measures within each group and between the groups. In the case where analysis of variance illustrated a significant difference, the post hoc analysis with Tukey test was used for further comparison. Infarct size was analyzed using student The data presented demonstrate that Ex-4-mediated protection of the heart against I/R injury when infused at reperfusion is GLP-1-receptor-dependent. In contrast, GLP-1 (9-36) protection under the same conditions appears to be GLP-1-receptor-independent. These findings highlight the need for a greater understanding of the mechanisms of GLP-1 (9-36)’s GLP-1-receptor independence with the aim of translating these findings to the clinic .


Downregulation of epithelial markers and upregulation of mesenchymal markers are the characteristics of the epithelial to mesenchymal transition (EMT) program, which provides the metastatic advantage of breast cancer. However, the mechanism underlying the switch of EMT markers remains poorly understood.In this study, we used the affinity purification and mass spectrometry coupled approach to identify the interactome of Slug. CoIP, GST-pulldown, ChIP, Re-ChIP, qPCR and Immunoblot were used to investigate the underlying mechanism of Slug-PRMT5-LSD1 complex. The role of PRMT5 and LSD1 in breast cancer progression was evaluated both in vivo and in vitro.Here we found that the transcription factor Slug associates with PRMT5 and LSD1 in a complex and facilitates the breast cancer invasion in vitro. Mechanistically, PRMT5 and LSD1 work with Slug to exert dual transcriptional activities to inhibit E-cadherin expression by PRMT5-catalyzed H4R3me2s and LSD1-mediated demethylation of H3K4me2 on the E-cadherin (CDH1) promoter, and activate vimentin (VIM) expression via PRMT5-driven H3R2me2s and LSD1-mediated removal of H3K9me2. Importantly, PRMT5 and LSD1 are coordinately expressed in breast cancer patients and pharmacologic perturbation of both PRMT5 and LSD1 shows a synergetic effect on the inhibition of breast tumor growth and metastasis in vivo.Our study suggests that PRMT5 and LSD1 function as a dual epigenetic modifier to promote Slug induced EMT program, suggesting that the inhibition of PRMT5 and LSD1 presents a potential therapeutic strategy against cancer metastasis.The online version contains supplementary material available at 10.1186/s13046-022-02400-7. Accumulating evidences have demonstrated that the epithelial to mesenchymal transition (EMT) process plays a crucial role during malignant tumor metastasis, and is associated with generation of drug resistance and cancer stem cell. During the EMT, epithelial cells shed their epithelial characteristics, including loss of cell junctions and apical-basal polarity, reduction of epithelial markers, followed by the gain of mesenchymal properties including enhanced invasiveness, reorganization of cytoskeleton and increased expression of mesenchymal markers. EMT is tightly orchestrated by EMT-inducing transcription factors  that alter gene expression to trigger the epithelial to mesenchymal phenotype conversion [Slug , a member of the SNAIL superfamily of zinc-finger transcription factors, plays a pivotal role in modulating the expression of genes responsible for the EMT during embryogenesis and cancer . PreviouOther chromatin modifiers may be involved in assisting Slug. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase, which is known to carry out symmetrical dimethylation on histone substrates  and non-histone proteins. PRMT5 is involved in gene silencing through the induction of repressive histone markers such as symmetrical dimethylation of H4R3 and H3R8, whereas PRMT5-catalyzed H3R2 symmetric dimethylation has been shown to mediate transcriptional activation . PRMT5-mIt has been reported that Lysine-specific demethylase 1 (LSD1) associates with Snail and Slug, and is essential for Snail and Slug-mediated EMT and transcriptional inhibition of epithelial genes , 17. LSDAntibodies used include: anti-Slug , anti-LSD1 , anti-myc , anti-HA  from Cell Signaling Technology; anti-PRMT5 , anti-H4R3me2s , anti-H3K9me2  from Abcam; anti-GAPDH , anti-H3 , anti-H4  from Proteintech; anti-E-cadherin , anti-vimentin  from BD Biosciences; anti-Flag  from PROSPEC; anti-H3R2me2s  from Epigentek; anti-H3K4me2  from Millipore; anti-Slug  from Santa cruz. Anti-Flag M2 affinity gel (A2220) and Flag peptide (F3290) were purchased from Sigma. SP2509 (HY-12635) and EPZ01566 (HY-12727) were obtained from MCE. The cDNA for wild-type or deletion mutants of Slug, PRMT5 and LSD1 was amplified by PCR and cloned into EcoRI site of a lentiviral vector CD532A with Flag, myc or HA tag, respectively or a bacterial expression vector pGEX-KG by homologous recombination using ClonExpress® II One Step Cloning Kit . The shRNA sequences targeting human Slug, PRMT5 and LSD1 were synthesized by RuiBiotech and subcloned into a pLKO.1 vector (Addgene). For knockdown experiments, two independent shRNA sequences were targeted for each gene and the one showing the higher silencing efficiency was used for the subsequent experiment. All DNA sequences were verified by sequencing. The shRNA targeting sequences used were: shSlug-1#, GAACTGGACACACATACAGTG; shSlug-2#, CAGACCCATTCTGATGTAAAG; shPRMT5-1#, GCCCAGTTTGAGATGCCTTAT; shPRMT5-2#, GCGTTTCAAGAGGGAGTTCAT; shLSD1-1#, CCACGAGTCAAACCTTTATTT; shLSD1-2#, GCTACATCTTACCTTAGTCAT.HEK293T, MCF10A and MDA-MB-231 cell lines were purchased from the American Type Culture Collection . HEK293T and MDA-MB-231 cells were maintained in DMEM (Thermo Scientific™) plus 10% fetal bovine serum (FBS) (ExCell Bio) and penicillin, streptomycin (Thermo Scientific™). MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum (Gibco), 20 ng/mL EGF (R&D), 0.5 mg/mL hydrocortisone , 100 ng/mL cholera toxin (Sigma), 10 mg/mL insulin (MCE) and penicillin/streptomycin. SUM159 cell line was purchased from Meisen Chinese Tissue Culture Collection  and grown in RPMI1640 (Thermo Scientific™) supplemented with 10% FBS and penicillin, streptomycin. All the cell lines are routinely checked for morphological and growth characteristics. Mycoplasma testing of cell cultures was performed routinely using a MycoBlue Mycoplasma Detector Kit .A lentiviral vector bearing the shRNA or cDNA of interest, along with a packing vector (psPAX2) and an envelope vector (pMD2.G) was co-transfected into HEK293T cells using polyethylenimine. Supernatants containing virus particles were collected at 48 h post transfection, and filtered through 0.45 μm filters to remove cell debris. The viruses were used to infect target cells grown in medium supplemented with 8 μg/mL polybrene. Infected cells were then obtained in the presence of 1 μg/mL puromycin.Cells were processed in lysis buffer  on ice for 1 h and centrifuged at 16,000 g for 10 min at 4 °C to collect the supernatant. After the protein concentrations were measured by the BCA protein assay kit (Thermo Scientific™), samples with equal amounts of proteins were mixed with loading buffer and boiled. Protein lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Then, the membranes were blocked in 5% non-fat milk for 1 h at room temperature, incubated with a primary antibody overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Subsequently, the membranes were probed with ECL reagent (Millipore) and proteins were visualized by a Tanon-5200 Automatic Chemiluminescence Imaging Analysis System .8 cells were incubated with 150 µL equilibrated anti-Flag M2-agarose beads for 1.5 h at 4 °C. After binding, the beads were washed with cold lysis buffer five times. Flag peptide (Sigma) was added into the resin to elute the Flag protein complex overnight at 4 °C and centrifuged at 8,000 g to collect the supernatant. Then the supernatant was resolved on 4–12% gradient gels (GenScript), stained using silver stain kit (Pierce), and subjected to LC–MS/MS (ThermoFisher Q Exactive mass spectrometer) sequencing.HEK293T cells expressing Flag-Slug were lysed in lysis buffer  on ice for 30 min and centrifuged at 10,000 g for 10 min at 4 °C to collect the supernatant. Cell lysates extracted from about 5 × 10Cell lysates were obtained by incubating the cells in lysis buffer  for 20 min at 4 °C, followed by centrifugation at 14,000 g for 15 min at 4 °C. Overall, 5% whole-cell extracts were used for input. The rest of the protein extracts were incubated with 2 μg control or specific antibodies overnight at 4 °C. Then, 10 μL of Protein G magnetic beads (Invitrogen) were added with further incubation at 4 °C for 2 h. Beads were then washed five times using the cold lysis buffer. The immunoprecipitates were boiled with 2 × SDS-PAGE loading buffer, separated on SDS-PAGE gels, followed by immunoblotting with various antibodies indicated.E. coli (BL21) and induced with 1 mM IPTG at 37 °C for 3 h. The cells were harvested by centrifugation at 4,000 rpm for 10 min. Then, the bacterial pellets were resuspended and lysed by sonication in cold PBS in the presence of complete protease inhibitor cocktail, followed by centrifugation to collect the supernatant at 12,000 g for 10 min at 4 °C. The cell lysates were applied to prepared glutathione-Sepharose 4B beads  and incubated for 2 h at 4 °C. Beads were washed five times using the cold PBS. Glutathione-Sepharose-bound GST fusion proteins were mixed with Flag-tagged fusion proteins, which were purified from HEK293T cells, and incubated for 2 h at 4 °C. The beads were then washed three times with wash buffer . The bound proteins were eluted by boiling in 2 × SDS-PAGE loading buffer, followed by immunoblotting analysis. The purified GST fusion proteins were examined for the presence by coomassie brilliant blue staining.GST fusion proteins were transformed in 3) with rotation at 37 °C for 30 min twice. Then the reverse crosslinking was carried out by adding NaCl (0.2 M) and proteinase K (0.5 mg/ml) and incubated at 65 °C overnight. DNAs were purified using a DNA purification kit. The purified DNA was dissolved in ddH2O for the qPCR. For Re-ChIP, immune complexes were eluted from the first IP by incubation with 10 mM DTT at 37 °C for 30 min. Eluents were diluted 1:50 in dilution buffer , and subjected to Re-IP with the secondary antibodies. DNA template enrichment was analyzed by conventional PCR. The following primers were used for the E-cadherin promoter: 5’-GAACCCTCAGCCAATCAGC-3’ (forward) and 5’- CTGACTTCCGCAAGCTCACA-3’ (reverse); vimentin promoter: 5’- GAGGGGACCCTCTTTCCTAA-3’ (forward) and 5’-GAGAGTGGCAGAGGACTGGA -3’ (reverse); Claudin-1 promoter: 5’- AGCTCTGGTGCCTGGTCCT-3’ (forward) and 5’- GGTTTCAGGGCGGCTCACC-3’ (reverse); ZEB1 promoter: 5’-GCTGCGCGGGTCAGGTAGC-3’ (forward) and 5’-GTCTGGACTCCCCGGGGAGT-3’ (reverse).Cells were cross-linked with 1% formaldehyde for 10 min at 37 °C and stopped by 125 mM glycine at room temperature for 5 min. Then cells were washed with cold PBS, and resuspended in cell lysis buffer  for 30 min at 4 °C. Samples were centrifuged to obtain the supernatant. The supernatant was subjected to sonication to shear the chromatin between 100 and 500 bp. After centrifugation at 16,000 g for 10 min at 4 °C, the protein-DNA complexes were immunoprecipitated with 2 μg control or specific antibodies overnight at 4 °C, followed by further incubation with protein G magnetic beads for 2 h at 4 °C. Complexes were washed with low-, high-salt and LiCl wash buffer sequentially, followed by two washes with TE buffer at 4 °C. The complex was eluted by adding 100 μl elution buffer  and then reverse transcribed into cDNA using TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix . RT-PCR was performed using 2 × Accurate Taq Master Mix . qPCR was performed using Taq Pro Universal SYBR qPCR Master Mix  on a QuantStudio 7 Flex Real-Time PCR system (ABI). GAPDH serves as an internal control in all experiments. The primer sequences used were as follows: E-cadherin: 5’- GACAACAAGCCCGAATT-3’ (forward) and 5’-GGAAACTCTCTCGGTCCA-3’ (reverse); vimentin: 5’- GAGAACTTTGCCGTTGAAGC-3’ (forward) and 5’-GCTTCCTGTAGGTGGCAATC-3’ (reverse); Slug: 5’-GGTCAAGAAGCATTTCAAC-3’ (forward) and 5’-GGTAATGTGTGGGTCCGA-3’ (reverse); PRMT5: 5’-GAGAATGCACCAACTACACA-3’ (forward) and 5’-ATTTCAAGAGCCACTGCAAT-3’ (reverse); LSD1: 5’-GAATTTGCTAATGCCACACC-3’ (forward) and 5’-GTATTCACAGCTATCACTTCAC-3’ (reverse); GAPDH: 5’-ATGACCCCTTCATTGACCTCA-3’ (forward) and 5’-GAGATGATGACCCTTTTGGCT-3’ (reverse); Claudin-1: 5’-TATTTCTTCTTGCAGGTCTGGCT-3’ (forward) and 5’-CTGGCATTGACTGGGGTCAT-3’ (reverse); ZEB1: 5’-TGCACTGAGTGTGGAAAAGC-3’ (forward) and 5’-TGGTGATGCTGAAAGAGACG-3’ (reverse).Immunohistochemical staining was carried out to assay PRMT5 or LSD1 on the breast cancer tissue microarray . Specifically, the tissue sections were deparaffinized and rehydrated by heating the sample at 95 °C in Tris–EDTA buffer (pH 9.0) for 20 min. Endogenous peroxidase activity was blocked by peroxidase . The sections were blocked with goat serum and then mixed with anti-PRMT5  or anti-LSD1 antibody  at 4 °C overnight, followed by mixing secondary antibodies  for 1 h and developed with 3.3′-diaminobenzidine. Hematoxylin was used to counterstain the nuclei. Samples were scored by the H-score method that combines the intensity of staining and the percentage of positive cells. PRMT5 or LSD1 levels were scored on the following scale: Staining Intensity (SI): 0, no staining; 1, weak staining; 2, moderate staining; 3, intensive staining; Percentage of Positive Cells (PP): 0, no positive cells; 1, less than 10%; 2, 11–50%; 3, 51–80%; 4, over 80%. The immune reactive score (IRS): SI × PP (0–12). IRS < 4 was considered as negative or weak staining (Low), 6–8 as moderate staining (Middle) and 9–12 as strong staining (High). Scoring of the breast cancer samples was done in a blind manner by a board-certified pathologist.4 cells per well into Matrigel-coated invasion chambers . The lower chambers contained culture media containing 10% FBS. The wells were washed with PBS and fixed with 4% paraformaldehyde. The cells on the apical side of each insert were removed by scraping. Cells migrated to the basal side of the membrane were stained with 0.1% crystal violet and processed using Image-pro plus software.The cells starved with DMEM medium supplemented with serum free media overnight were seeded at a density of 5 × 106 cells) were injected into the lateral tail vein for experimental metastasis model or fourth mammary pad for spontaneous metastasis model of 4-week-old female nude mice, respectively. The mice were divided randomly into 4 groups including Vehicle, SP2509 , EPZ015666  and the combination of SP2509 and EZP015666 (five mice per group). The drugs were subsequently administered by intraperitoneal (i.p.) injection on days 10, 13, and 17 in an eight days repeating cycle for three cycles. The growth of primary tumors in mice that inoculated with cancer cells via fourth mammary fat pad injection were measured with a vernier caliper. The tumor volume was determined using the formula volume = (length) × (width)2 × 0.5. For the spontaneous metastasis assay, the lungs were removed and imaged using an IVIS Lumina II imaging system  at day 41 post orthotopic transplantation. To examine for seeding metastases, mice that obtained cancer cells via intravenous injection were imaged at day 41 post tail veil injection by an IVIS Lumina II imaging system, then the lungs were removed and imaged using an IVIS Lumina II imaging system as well. All animal experiments were approved by the Animal Care Committee of Southern University of Science and Technology.MDA-MB-231 cells that had been engineered to express firefly luciferase stably (1 × 10https://www.ncbi.nlm.nih.gov/geo/), using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trim-galore  to quality control and filtering for the sequencing reads. The sequencing reads were mapped to the reference genome  through bowtie2 (version 2.4.5). The samtools were used for transforming the Sam format file to the Bam format file and to sort the Bam file. Using the samtools to remove the PCR duplicates, then Macs2 was applied for calculating the peak value of the ChIP-seq datasets to compare the treatment IP DNA and the control input DNA. A significant threshold of 10–2 was applied to all datasets. R-package ChIPseeker was used to annotate the peaks. We used MEME-ChIP for motif discovery. Using bamCoverage, we convert the bam file to the bw file. With the IGV genome browser, we visualize the peaks. The pie plot was drawn by the ggplot2 package and the Venn diagram was drawn by the Venn diagram package. All these packages were installed in the R (4.0.3).Three ChIP-seq raw datasets  were derived from the GEO database (Wild-type (CCCAAA) or mutated (TCTGAG) E-cadherin or vimentin promoter sequence (-500 bp to TSS) was cloned into the firefly luciferase reporter pGL3-basic-based vector by homologous recombination with a ClonExpress® II One Step Cloning Kit . Next, HEK293T cells were co-transfected with luciferase reporter, pRL-TK Renilla luciferase vector and indicated expression constructs. After 48 h, the luciferase activity of total cell lysates was assayed using a Dual-Luciferase Reporter Assay System (Promega). Data was normalized against Renilla luciferase activity.Biotin-labeled double-stranded wild-type (CCCAAA) or mutated (TCTGAG) oligonucleotides or corresponding nonbiotinylated oligonucleotides were incubated with the Slug-PRMT5-LSD1 complex, which was prepared from HEK293T cells co-transfected with Flag-Slug, HA-PRMT5 and HA-LSD1 at 4 °C overnight under gentle rotation. The biotinylated DNA/protein complexes were captured using magnetic streptavidin beads at 4 °C for 2 h followed by three washes. Subsequently, bound proteins were detected by immunoblotting. Oligonucleotide sequences used were as follows: E-cadherin (wt): CAAAACGAACAAACAAAAAATCCCAAAAAACAAAAGAACTCAGCCAAGTG; vimentin (wt): TCAGACTATCATCCGGAAAGCCCCCAAAAGTCCCAGCCCAGCGCTGAAGT; E-cadherin (mut): CAAAACGAACAAACAAAAAATTCTGAG AAACAAAAGAACTCAGCCAAGTG; vimentin (mut): TCAGACTATCATCCGGAAAGCCTCTGAGAGTCCCAGCCCAGCGCTGAAGT;2 test. In the statistical analysis, P < 0.05 is deemed as statistically significant.The data represent the mean ± SD values of samples obtained from three independent experiments. We performed a two-tailed Student’s t test to determine statistically significant differences between two groups. Differences between multiple groups were determined by Dunnett’s multiple comparison test. The Kaplan–Meier method was employed to plot survival curves, and differences were calculated with the log-rank test. The relationship between PRMT5 and LSD1 expression in human breast cancer samples were evaluated by χTo gain the mechanistic insight into the Slug mediated transcriptional regulation, we used the affinity purification and mass spectrometry coupled approach to survey the interactome of Slug. Whole cell extracts were prepared from HEK293T cells expressing Flag-Slug and subjected to purification using an anti-Flag affinity gel. Mass spectrometric analysis indicates that Slug immunoprecipitates contain LSD1 and PRMT5  of endogenous Slug from MDA-MB-231, SUM159 and Hs578T cells brought down LSD1 and PRMT5, suggesting that Slug interacts with these proteins  includes the SNAG domain of Slug, and the C-terminal Slug (128–268 aa) contains the conserved zinc finger motif , the middle Rossmann-fold segment (304–460 aa) and the C-terminal β-barrel domain (461–637 aa) Fig. A. To ideThe N-terminal region of LSD1 comprises a SWIRM domain 1–276 aa) and the larger C-terminal segment of LSD1 includes a catalytic amine oxidase (AO) domain (277–876 aa)  was applied to uncover various cellular events for the genes corresponding to these co-occupied promoters. These biological processes include cytoskeleton organization, cell morphogenesis, metabolism and development , PRMT5 GSE130194) and LSD1 (GSE101150). We identified 7136 LSD1-specific binding peaks, 19,628 PRMT5-specific binding sites, and 73,702 Slug-specific binding sequences mostly residing in the promoter, intronic or intergenic regions  and vimentin (VIM) Fig. D. AnalysWe then evaluated the regulation of E-cadherin and vimentin by the Slug-PRMT5-LSD1 complex. We found that Slug, PRMT5 and LSD1 co-occupied the promoters of E-cadherin and vimentin through the ChIP assays using antibodies against Slug, PRMT5 or LSD1 in MDA-MB-231 cells Fig. A. These To further confirm the transcription regulation of E-cadherin/vimentin by the Slug/PRMT5/LSD1 complex, we performed dual-luciferase reporter assay. As shown, Slug was able to repress the E-cadherin (WT) or activate vimentin (WT) promoter activity , the EPZ015666 (100 mg/kg) group, the SP2509 plus EPZ015666 group. Tumor volumes were measured at indicated time with calipers and harvested on day 41. Monotherapy with either SP2509 or EPZ015666 alone partially inhibited the growth of the breast tumors, interestingly, the combined addition of SP2509 and EPZ015666 caused obvious synergistic effects in reducing tumor volumes F. In addin = 1222, P < 0.01, Fig. http://kmplot.com/analysis/) demonstrated that both enhanced LSD1 expression and higher PRMT5 expression were associated with shorter relapse-free survival (RFS) and overall survival (OS) of breast cancer patients  database to compare PRMT5 or LSD1 expression in normal human breast tissues and breast cancer specimens. We found that breast cancer samples expressed significantly higher PRMT5 or LSD1 levels than normal breast cells,  analysis. Interestingly, the expression of PRMT5 positively correlated with LSD1 in breast tumor specimens Fig. C, D. To We further analyzed the PRMT5 and LSD1 co-expression in distinct breast cancer subtypes. We found that PRMT5 expression was positively correlated with the expression of LSD1 in both mRNA and protein level among all subtypes including luminal, HER2 + and Basal-like subtype Figure SA, B. IntIn this study, we report that Slug, PRMT5 and LSD1 interact and work together to modulate the expression of E-cadherin and vimentin that are the molecular markers of EMT. Slug has been documented as both an activator and a repressor of transcription on different target genes involved in EMT program –12. The PRMT5 is known to interact with several transcription complexes to formulate a transcription supression program to promote EMT process. Previous studies identified PRMT5 as a suppressor recruited to the Snail complex through the interplay with NuRD (MTA1) or AJUBA corepressor to inhibit the expression of a group of genes such as E-cadherin and α-catenin , 24. PRMLSD1 has been suggested to coordinate with EMT transcription factors and chromatin-modifying enzymes to act as a transcription repressor to trigger the EMT program. It is well documented that Snail brings the LSD1-CoREST complex to the E-cadherin promoter for transcriptional repression by catalyzing the demethylation of H3K4me2 . This isOur study indicates that PRMT5 and LSD1 cooperate together with Slug to exert transcriptional activation via H3K9me2 demethylation and H3R2me2s on the vimentin promoter, as well as transcriptional repression through H3K4me2 erasure and H4R3me2s on the E-cadherin promoter. Covalent histone modifications are generally associated with either repression or activation of transcription and play an important role in carcinogenesis , 33. H3RQuite a few transcription regulators work in similar fashion to modulate gene expression. Another EMT inducer Twist1 has been found to interact with SET8 to participate in transcriptional repression and activation on the promoters of the Twist1 target genes E-cadherin and N-cadherin via catalyzing H4K20 monomethylation . EarlierTumor metastasis is the major cause of death in cancer patients. Metastatic breast cancer is generally considered intractable. EMT has been well studied to be a vital trigger of cancer metastasis and correlated with the generation of cancer stem cells and drug resistance, which makes it an attractive therapeutic target for metastatic cancer patients . EMT traIn this study, we found that Slug works with PRMT5 and LSD1 to act as a dual epigenetic regulator on the promoters of E-cadherin and vimentin. We demonstrated that PRMT5 and LSD1 cooperate to promote the EMT and invasion of cancer cells, and that PRMT5 expression is positively correlated with LSD1 expression in breast carcinoma specimens. Our data suggest that the combination of LSD1 inhibitor (SP2509) and PRMT5 inhibitor (EPZ015666) synergistically lead to a strong reduction of vimentin expression, induction of E-cadherin expression and decreased invasion of breast cancer cells in vitro, and also efficiently inhibit tumor growth and lung metastases of breast cancer in vivo. Our findings provide a promising therapeutic strategy for the treatment of patients with metastatic breast cancer.Additional File 1.