Ashes from sugarcane by-product incineration were used to synthesize silica powders through alkaline hot extraction, followed by ethanol/acid precipitation or the sol–gel method. Both production methods allowed amorphous spherical silica microparticles with sizes ranging from 1–15 μm and 97% purity to be obtained. Water absorption ranged from 135–155 mL/100 g and 150–250 mL/100 g for precipitated silica and silica gel, respectively, while oil absorption ranged from 305 to 390 and from 250 to 350 mL/100 g. The precipitation with ethanol allowed the recovery of 178 g silica/kg ash, with a lab process cost of EUR 28.95/kg, while the sol-gel process showed a yield of 198 g silica/kg ash with a cost of EUR 10.89/kg. The experimental data suggest that ash from sugarcane by-products is a promising source to be converted into a competitive value-added product, minimizing the environmental impact of disposal problems. In Brazil and India, the sugarcane industry generates more than 800 million metric tons of sugarcane waste per year, particularly straw and bagasse . This wa2) content, above 60 g/100 g ash, in crystalline and amorphous phases, such as quartz and cristobalite. The primary source of silica is the silicic acid available in the soil, which is absorbed by the sugarcane roots and transported to the leaves, where it is deposited as amorphous silica [Sugarcane ash has high silica , obtained through the incineration of sugarcane bagasse, was provided by Raízen . The raw material was sieved, and the fraction with a particle size above 160 μm was used for the extraction of biogenic silica. Absolute ethanol (p.a > 99.8%) and sulfuric acid (p.a 95.0–97.0%) were purchased from Honeywell , and sodium hydroxide pellets were purchased from LabChem . Nitric acid 65% (Suprapur) and fluoric acid 40% were acquired from Merck KGaA . Commercial silica Spheron L-1500 was a gift from Presperse .SCBA was characterized for total ashes, moisture content, and total organic matter following the Association of Official Analytical Chemists (AOAC) methods ,16.3 65% and 2 mL of HF 40% and heated in a microwave system . The digestion procedure was conducted in three steps: 140 °C and 40 bar for 5 min; 160 °C and 40 bar for 10 min; 200 °C and 40 bar for 30 min; and 100 °C and 20 bar for 5 min. After microwave digestion, the samples were cooled down to room temperature and diluted to a final volume of 50 mL. Microwave-digested samples were then analyzed through ICP–AES  and quantified through external standard calibration. All the analyses were performed in triplicate.Silica and other mineral contents were determined by Inductively Coupled Plasma–Atomic Emission Spectrometry (ICP–AES). Briefly, 200 mg of SCBA were mixed in a Teflon vessel with 7 mL of HNO2O of 1:10), and the mixture was thoroughly stirred and filtered through a paper filter. The solid fraction was discarded, and the liquid fraction, rich in sodium silicate, was stored at room temperature.Nickel-plated stainless steel crucibles were used to mix ash and sodium hydroxide (SCBA: NaOH pellets mass ratio of 1:2), and the mixture was placed into a muffle oven at 350 °C for 20 min. After the removal of the crucibles, deionized water was cautiously added (mass ratio SCBA: H2SiO3 solution was adjusted to 6 using H2SO4 10% (v/v) under agitation. The solution was then filtered under vacuum (pressure 300–400 mbar) at room temperature using a cloth filter and washed with deionized water using a mass ratio of gel solution: water of 1:3. The final gel was resuspended in water, the viscosity was adjusted to 10–12 cP using a Vibro Viscometer SV-10 , and the solution was fed into a Niro Atomizer Mobile Minor Spray-Dryer using an inlet temperature of 180 °C and a pump flow of 2.5 L/h.For the production of silica gel microparticles, the pH of Na2SiO3 solution (volume ratio 1:1), and the pH was adjusted to 8.5 using H2SO4 10% (v/v) under agitation. The solution was then filtered under vacuum (pressure 300–400 mbar) at room temperature using a cloth filter and washed with deionized water using a mass ratio of gel solution: water of 1:3. The final gel was dried overnight at 105 °C. The powder was ground, resuspended in deionized water at 20% (w/w), and stirred overnight at 50 °C. The final solution was fed into a Buchi B-191 Mini spray dryer. The operating conditions were as follows: inlet temperature, 160 °C; outlet temperature, 100–110 °C; gas flow, 600 L/h; pump rate, 0.23 L/h; aspirator rate, 25.5 m3/h.For the production of precipitated silica microparticles, absolute ethanol was added to the Na3 65% and 2 mL of HF 40% and heated in a microwave system . The digestion procedure was conducted at 185 °C and 35 bar for 70 min. Microwave-digested samples were then analyzed through ICP–AES  and quantified through internal standard calibration. All analyses were performed in triplicate.Briefly, 200 mg of biogenic silica were mixed in a Teflon vessel with 2 mL of HNOPowder X-ray Diffraction Analysis (PXRD) was performed on a Rigaku MiniFlex 600 diffractometer with Cu kα radiation with a voltage of 40 kV and a current of 15 mA .−1 with a resolution of 1 cm−1.Attenuated Total Reflection–Fourier Transform Infrared Spectroscopy (ATR–FTIR) analysis was conducted using a Spectrum 100 FTIR spectrometer  equipped with an ATR accessory  with a diamond/Se crystal plate. The number of scans was set to 64, and spectra were collected in the absorbance range of 4000 to 400 cmSEM microparticle analyses were performed on a JEOL-5600 LV Scanning Electron Microscope  from JEOL, Japan, equipped with a SPRITE HR Four Axis Stage controller . Samples were placed on metallic stubs covered with adhesive carbon tape  and coated with gold/palladium using a Sputter Coater . All observations were performed under high vacuum with an acceleration voltage of 30 kV at a working distance of 12–13 mm and a spot size of 20.10, D50, and D90) was size at which 10, 50, and 90% of the particles were under the average particle size and span (SPAN = (D90 − D10)/D50).Particle size distribution was analyzed in a Coulter LS 230 laser granulometer  equipped with a small volume module. The measurement was made in water, using the Fraunhofer laser diffraction method. All the analyses were performed in duplicate. The particle size distribution .The obtained results were analyzed with TIBCO Data Science Workbench (Statistica) v 14.0.0.15 using one-way analysis of variance (ANOVA) with the Tukey HSD post hoc test. The differences were considered significant at a level of 95% (p > 0.005). With silica present in significantly higher concentrations than other minerals (p < 0.01), SCBA proved to be an efficient source of this mineral. In fact, sugarcane crops can take up between 500 and 700 kg Si.ha−1, while rice husk, a more studied silicon source, absorbs between 230 and 470 kg Si.ha−1 [Sugarcane bagasse ashes presented 5.58 ± 0.05 g/100 g of moisture content, 82.28 ± 0.37 g/100 g of minerals, and 12.14 ± 0.16 g/100 g of organic matter. The minerals present in SCBA and their concentration are presented in  Si.ha−1 . Neverth Si.ha−1 . This crThe production of biogenic silica allowed smooth white powders with a purity of ca. 97.3% and similar chemical compositions to be obtained. Minor contaminants included sodium 1.40 ± 0.23 g/100 g), aluminum (0.59 ± 0.02 g/100 g), iron (0.16 ± 0.041 g/100 g), zirconium (801 ± 41 ppm), magnesium (363 ± 16 ppm), chromium 136 ± 22 ppm), manganese (142 ± 5.5 ppm), zinc (83.5 ± 2.6 ppm), phosphorous (54.9 ± 19.9 ppm), copper (21.1 ± 1.2 ppm), barium (21.3 ± 0.2 ppm), and nickel (5.26 ± 1.08 ppm). Some of these impurities have origins in SCBA, such as potassium, calcium, magnesium, and iron, in accordance with the characterization of several authors for other ashes, such as those from sugarcane, rice husks, and bamboo leaves ,22,23. O ppm, man g/100 g,The greatest silica yield was obtained for the silica gel process, 198 g silica/kg SCBA, while the precipitated silica microparticles process provided a yield of 178 g silica/kg SCBA. These silica extraction yields were much lower than others reported for similar biomasses, ranging from 45 to 90% for sugarcane or rice husk ashes, mainly due to the high loss of biomass during the sieving process, which allows the production of 100% biogenic silica ,22,25.The Powder X-ray diffraction patterns of the precipitated silica gel powder and the commercial silica powder Spheron L-1500, an amorphous silica benchmark widely used in cosmetic formulations, are depicted in −1 for both prototypes and the commercial benchmark, corresponding to the overlapping of the O–H stretching bands of hydrogen-bonded water molecules and the SiO-H stretching of hydrogen-bonded surface silanols [−1 corresponding to the deformation vibrations of the adsorbed water, which is common in silica aerogels due to the presence of surface silanol groups and was confirmed by the presence of a sharp peak at 996 cm−1, which provided higher hydrophilicity compared with other types of silica [−1 represented the asymmetric stretching vibrations of siloxane covalent bonds (Si-O-Si), which shifted to 1025 cm−1 for precipitated silica, while the peak at 887 cm−1 represented the symmetric stretching vibration of the same group, characteristic of the silica network.The analysis of the FTIR spectra  revealedsilanols . It was f silica . The larThe particle shape is critical to achieving the desired functional performance of silica, as non-spherical silica particles do not allow silky powder to be obtained and significantly increase the oil absorption capacity, leading to a dry sensation when applied to the skin. Scanning Electron Microscope (SEM) images of silica powders demonstrate the clear spherical appearance of silica microparticles produced following both methodologies . SpheronBesides the particle shape, the size of silica particles also affects their technical performance, including the oil and water absorption capacity. With both methods, it was possible to obtain a silica powder without the presence of nanoparticles (<1000 nm), ensuring that it was suitable for cosmetic application. According to Scientific Committee on Consumer Safety (SCCS) guidance on nanomaterials in cosmetics, there is a concern related to the use of nanosilica in cosmetic formulations, as they might be able to permeate the skin barrier and be carried by the bloodstream and accumulate in the internal organs, which could potentially lead to toxicity .p < 0.05) oil absorption capacity, 289.7 ± 23.4 g sunflower oil/100 g silica, than precipitated silica, which presented an oil absorption of 332.9 ± 11.7 g sunflower oil/100 g silica. Both synthesized prototypes presented an oil absorption capacity significantly higher than the cosmetic benchmark Spheron L-1500 (p < 0.01). These results could be explained by the lower interparticle porosity (0.159) of Spheron L-1500 compared with silica gel, with an interparticle porosity of 0.448, and compared with precipitated silica, with an even higher interparticle porosity of 1.120 (data not shown).Undesirable skin shine is composed of sebum, sweat, dead skin cells, and environmental impurities. Thus, an ingredient that presents both humidity and the ability to absorb sweat and oil and remain dry after being absorbed, will have a better mattifying effect than ingredients that mainly absorb oil or that become wet and cakey after absorbing moisture . Sphericp < 0.05) water absorption capacity, of 169.4 ± 10.4 mL/100 g, while precipitated silica presented a water absorption of 145.0 ± 7.1 g/100 g. By contrast, both sugarcane-derived silica prototypes presented a significantly lower (p < 0.01) water absorption capacity than Spheron L-1500, which was 262.0 ± 7.5. These water absorption values are, however, far higher than the water absorption reported by Chindaprasirt and Rattanasak [2/kg, data not shown), which is a well-known moisture absorber widely used in different products and applications as a desiccant. Amorphous silica comprises an inorganic network of SiO2 units bonded through hydrophobic siloxane groups and holding hydrophilic surface silanol (Si-OH) groups [These results are in accordance with several authors, who achieved oil absorption values between 150 and 380 g oil/100 g silica aerogel ,32,33 an) groups .A comprehensive analysis of economic viability is needed to predict the feasibility of large-scale production of biogenic silica; therefore, both lab processes’ mass balances, presented in This difference is explained by the higher consumption of chemicals in the production of precipitated silica, in particular the use of absolute ethanol, which represented an increase in chemical costs, from 29% (EUR 3.18/kg) for silica gel to almost 36% (EUR 10.30/kg) in precipitated silica. Regarding the energy costs, they were also expected to be higher in precipitated silica, as this process requires an additional operation unit—an oven. Thus, the total energy cost for silica gel was EUR 2.87/kg, with the heating reactor and spray-dryer units being the most expensive steps, while the energy cost for precipitated silica was EUR 5.79/kg, with the oven step portraying 45% of energy costs.To make this economic simulation as realistic as possible, the costs for chemicals and energy were based on bulk quantity prices, although the mass balances were determined at the lab scale. Lab scale processes are usually more prone to lower yields, whether by the mass losses along the process or the reduced yield of some lab equipment, such as the spray dryer.Two prototypes of biogenic amorphous silica were synthesized from sugarcane bagasse ashes— precipitated and silica gel microparticles—with a purity of up to 98%. Their microstructure was composed of mesoporous spherical particles with sizes between 1 and 15 μm, providing optimal oil and moisture absorption capacities. Associated with the lack of nanometric particles and heavy and potential sensitizer metals, these prototypes are safe and ideal for cosmetic applications. Moreover, a process development study and cost analysis allowed the optimization of the process, guaranteeing the economic viability of silica as a commercial ingredient. These prototypes represent a breakthrough in the cosmetic market that lacks sustainable and biogenic options.


Knockdown of YAP in primary chondrosarcoma cells significantly inhibited chondrosarcoma proliferation, invasion, and tumorsphere formation. Chondrocyte lineage ablation of YAP delayed chondrosarcoma progression and lung metastasis in Col2-Cre;Trp53f/f/Rb1f/f mice. Moreover, we found that metformin served as a YAP inhibitor, which bound to the activity area of YAP protein, and inhibited chondrosarcoma cell proliferation, migration, invasion, and progression in vitro and significantly suppressed chondrosarcoma formation in vivo. Collectively, this study identifies the inhibition of YAP may be an effective therapeutic strategy for the treatment of chondrosarcoma.Chondrosarcoma (CHS) is a rare type of soft sarcoma with increased production of cartilage matrix arising from soft bone tissues. Currently, surgical resection is the primary clinical treatment for chondrosarcoma due to the poor response to radiotherapy and chemotherapy. However, the therapeutic effect is not satisfactory due to the higher local recurrence rate. Thus, management and elucidation of the pathological mechanism of chondrosarcoma remain an ongoing challenge, and the development of effective chondrosarcoma mouse models and treatment options are urgently needed. Here, we generated a new transgenic chondrosarcoma model by double conditional deletions of Trp53 and Rb1 in chondrocyte lineage which spontaneously caused spinal chondrosarcoma and lung metastasis. Bioinformatic analysis of the human soft sarcoma database showed that Trp53 and Rb1 genes had higher mutations, reaching up to approximately 33.5% and 8.7%, respectively. Additionally, Trp53 and Rb1 signatures were decreased in the human and mouse chondrosarcoma tissues. Mechanistically, we found that YAP expression and activity were significantly increased in mouse Col2-Cre;Trp53 Chondrosarcoma is a rare type of primary bone cartilage malignancy with an incident rate of about two new cases per a million populations per year , 2. It iTo discover a new drug or for drug repurposing, developing a mouse model that closely mimics human chondrosarcoma initiation and progression is one of the most important approaches in the clinic. Currently, only a few chondrosarcoma animal models have been developed including allograft tumor transplanted into the hamster or rat , 8. DespThe identification of some tumor suppressors’ mutations in cancer tissues can provide new strategies and options for the development of the new drug targets. Among these tumor suppressors, the mutations of Trp53 and Rb1 are most well-studied in different tumors. Notably, alterations of Trp53 and Rb1 were observed in about 33-96% and 20-50% of chondrosarcomas, respectively –16. AddiHippo pathway is crucial for skeletal development and tumorigenesis through modulating the activity of the essential downstream effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) –21. The f/f/Rb1f/f chondrosarcoma mouse model. Thus, this study provides a new transgenic chondrosarcoma model and a proof of principle that the inhibition of YAP activity may be a potential therapeutic target for chondrosarcoma.Here, we explored the function and molecular mechanisms of Trp53 and Rb1 in driving chondrosarcoma formation and lung metastasis. Our data showed that double deletions of Trp53 and Rb1 in chondrocytes led to the spontaneous development of chondrosarcoma through activation of YAP signaling. Deletion of YAP or inhibition of YAP by metformin significantly inhibited the chondrosarcoma progression in this new Col2-Cre;Trp53To understand the function of Trp53 and Rb1 in chondrosarcoma formation, we first analyzed the mutation rates of some tumor suppressors in the soft sarcoma database from TCGA (The Cancer Genome Atlas). Encouragingly, our data showed that Trp53 and Rb1 had higher mutation rates reaching up to 33.5 and 8.7%, respectively Fig. . Furtherf/f, Rb1f/f and Trp53f/f/Rb1f/f floxed mice with a transgenic Cre line driven by a Col2a1 promoter . qRT-PCR data verified that Trp53 and Rb1 were abrogated in chondrocytes , we constructed a triple conditional knockout mouse model Col2-Cre;Trp53f/f/Rb1f/f/YAPf/f (triple cKO mice). As expected, our X-ray data showed that deletion of YAP could partly protect against the spinal bone destruction and display a decreased volume of chondrosarcoma in Col2-Cre;Trp53f/f/Rb1f/f mice, suggesting that inactivation of YAP in Col2-Cre;Trp53f/f/Rb1f/f mice could block chondrosarcoma progression . Trp53f/f/Rb1f/f mice were a gift from Dr. David M. Feldser’s lab at the Department of Cancer Biology, University of Pennsylvania. The human chondrosarcoma and normal cartilage samples were purchased from the US Biomax company .Col2-Cre, and YAP® HD) were obtained from Promega Corporation.Trp53 (1C12), YAP (D8H1X), GAPDH antibodies, and Hippo Signaling Antibody Sampler Kit were purchased from Cell Signaling Technology. Antibody against Rb1 was purchased from Santa Cruz Biotechnology. The secondary fluorescent antibodies and H&E staining kit were from Abcam. DAB Substrate Kit was ordered from Vector Laboratories Inc. Plasmids pRL-TK, 8xGTIIC-luciferase, and shYAP1/2 were obtained from Addgene. The transfection reagents  at 37 °C for 30 min. Subsequently, these cells after digestion were harvested and cultured in α-MEM  supplemented with 10% FBS  and 1% Pen-Strep solution  at 37 °C with 5% humidified CO2, and the medium was replaced every other day. Micromass cultures were performed as previously described [Primary chondrocytes in this study were isolated from the embryonic limb buds of Col2-Cre;Trp53escribed .f/f, Col2-Cre;Rb1f/f, and Col2-Cre;Trp53f/f/Rb1f/f mice, or primary chondrosarcoma cells from Col2-Cre;Trp53f/f/Rb1f/f mice using TRIzol reagent  was reverse-transcribed into cDNA by PCR using PrimeScript™ RT Kit . qRT-PCR was then performed by CFX96 Real-Time PCR System and the SYBR Green mixture . GAPDH was served as an internal control and was determined by 2–∆∆Ct method. The primers of qRT-PCR in this study were listed in Supplemental Table Briefly, 1 μg total RNA was extracted from primary chondrocytes of Col2-Cre, Col2-Cre;Trp53Cell proliferation, migration, invasion, and tumorsphere assays were carried out as we performed previously , 49.For luciferase reporter assay, the chondrocytes were seeded and co-transfected with luciferase reporter and the indicated plasmids in the 12-well plate. After culturing for 48 hrs, the luciferase activities were analyzed by the Dual-Luciferase Assay Kit as we performed previously , 49.Radiographic procedures were performed in the Siemens X-ray equipment  as we performed previously , 49–51.f/f/Rb1f/f mice and controls were harvested, fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, decalcified with 14% EDTA in PBS (pH 7.4) for 1 month, and then embedded in paraffin. Soft tissues including the lung, kidney, spleen, brain, and liver from Col2-Cre;Trp53f/f/Rb1f/f mice and controls were fixed and embedded in paraffin. Six-micrometer sections of the above tissues were prepared, and then the stainings of Haemotoxylin and Eosin (H&E), Safranin O/fast green, and Alcian blue staining were conducted as we previously reported [Vertebrate bones from 5-month-old Col2-Cre;Trp53reported , 18, 50.f/f/Rb1f/f and Col2-Cre mice were seeded and cultured on coverslips. After a culture of 48 h, the coverslips were fixed in 4% PFA for 5 min at room temperature and then permeabilized with 0.3% Triton X-100 in PBS (PBST) 3 times per 5 min each. Next, the cells were blocked by 1% BSA for 1 hr at room temperature and probed with primary antibody against rabbit anti-YAP (1:200 dilution) overnight at 4 °C. After washing 3 times with PBST, the cells were incubated with Alexa Fluor® 594-conjugated second anti-rabbit antibody (1:1000 dilution) for 1 hr at dark. Then, a counterstain of nuclei was performed with DAPI and washed 3 times with PBST, and then the cells were visualized under a fluorescence microscope as we previously reported [For immunofluorescence staining, the primary chondrocytes from 5-month-old Col2-Cre;Trp53reported , 18, 50.reported , 50.f/f/Rb1f/f mice were harvested and lysed with RIPA lysis buffer and protein inhibitor cocktail , respectively. And then, an equal amount of the above proteins were subjected to SDS-PAGE gel , transferred to a PVDF membrane , and immunoblotted with primary antibodies YAP, pYAP, and GAPDH (1:1000 dilution) for overnight at 4 °C. Following 3 times washing with 0.1% TBST (Tween-20 in TBS), the PVDF membranes were incubated with HRP-conjugated anti-rabbit antibody (1:1000 dilution) for 1 hr at room temperature. After washing 3 times with TBST, the membranes were analyzed with ECL solution  as we previously reported [Briefly, the fresh chondrosarcoma tissues and adjacent normal cartilage from Col2-Cre;Trp53reported , 18, 50.f/f/Rb1f/f, Col2-Cre;Trp53f/f/Rb1f/f/YAPf/f, and Col2-Cre;Trp53f/f/Rb1f/f with the treatment of 400 mg/kg metformin mice and controls were measured using vernier calipers. And then, the chondrosarcoma volumes were calculated with the formula length × width2 × 0.5 [After euthanasia of the mice at indicated different time points, the width and length of chondrosarcomas from Col2-Cre;Trp53h2 × 0.5 , 53.p values <0.05 considered statistically. All data were downloaded and analyzed by R packages DESeq2 and ClusterPfofiler.The structures of YAP protein (6GE3) and metformin were obtained from the Protein Data Bank (PDB) and PubChem database of the National Center for Biotechnology Information (NCBI). The YAP-metformin docking was performed using PyMOL and AutoDock software. Public available human chondrosarcoma cells and normal chondrocytes database from Gene Expression Omnibus   were uset test. The statistical significance of multiple groups was determined by a two-way analysis of variance. P values <0.05 were considered significant.Experimental results were reported as mean ± SEM and analyzed by the software of SPSS 21, and the data were analyzed using Student’s SUPPLEMENTAL MATERIALvideochecklist


This work proposes an alternative method tomanufacture robust MXene-based structural materials for multifunctionalapplications.MXene-based structural materials with high mechanicalrobustnessand excellent electrical conductivity are highly desirable for multifunctionalapplications. The incorporation of macromolecular polymers has beenverified to be beneficial to alleviate the mechanical brittlenessof pristine MXene films. However, the intercalation of a large amountof insulating macromolecules inevitably compromises their electricalconductivity. Inspired by wood, short-chained hemicellulose (xylo-oligosaccharide)acts as a molecular binder to bind adjacent MXene nanosheets together;this work shows that this can significantly enhance the mechanicalproperties without introducing a large number of insulating phases.As a result, MXene–hemicellulose films can integrate a highelectrical conductivity (64,300 S m Hemicellulose(xylo-oligosaccharide) is a mixture of oligomerscomposed of xylose units connected by β-1,4 glycosidic bonds,normally including 2–7 branched or linear monosaccharide units. After self-assembly, hemicellulose withabundant oxygen-containing functional groups is embedded in orientedMXene nanosheets, tethering together adjacent nanosheets through hydrogenbonding (Table S3). Interestingly, the intercalation ofhygroscopic hemicellulose among conductive MXene layers provides moremoisture adsorption sites and promotes enlarging the interlayer spacing,endowing the composite film with a fast humidity response performance.As a result, the flexible MXene–hemi film can simultaneouslyintegrate a high mechanical strength (125 MPa), high electrical conductivity, high gravimetric capacitance (335F g–1), and multifunctionality when serving as astructural electrode with a very low hemicellulose content  via the HCl/LiF solution  peak located at 38.8°and the obvious shift of (002) from 9.5° to 8.5° after etching indicate the effective etchingAl layers in the MAX phase, which is consistent with previous works.32 After subsequent sonication, the obtained Ti3C2Tx show nearly transparent platelikemorphology with lateral sizes between several hundreds of nanometersand several micrometers . The morphology and dimension of Ti3C2Tx nanosheets are also proved by the AFM image. After the synthesis of Ti3C2Tx nanosheets, MXenefilms are fabricated by a vacuum-assisted filtration process. PristineMXene film shows a disordered and loosely lamellar structure witha rich interlayer space . Furthermore, we employed wide-angleX-ray diffraction (WAXD) characterization to evaluate the oritentionmore quantitatively.33 Specifically, thecharacterization was carried out at two directions . Notably, the MXene–hemi films shows a narrowerarc and full width at half-maximum (39.3°) compared to pristineMXene films, which indicates that MXene nanosheets in MXene–hemifilms are more preferentially oriented along the in-plane direction(f) was extracted from the azimuthalplots of the (002) peak (34 The orientation factorof MXene–hemi films (0.47) was slightly higher than that ofpristine MXene films (0.38), indicating an enhancement of the orientationof the microscopic structure after the incorporation of hemicellulose.35Tisolution 1a.28 Fro etchingS4 indica etchingS4 indica etchingS4 indica etchingS4 indica etchingS4 indicaFigure S8a).36 These polar groups playa vital role in interacting with hemicellulose. The deconvolutionof Ti 2p shows mixed moieties of Ti–C, Ti(II), Ti(III), TiO2, and Ti–C species  and Ti(III) aregradually increased (from 33% to 59%), while that of Ti–C isdecreased . These results demonstrate that the local chemical environmentsfor Ti–C of MXene have been changed for MXene–hemi films,forming more interactions with polar groups .In the case of O 1s spectra, a sharp increase of the C–Ti–OHpeak is consistent with Ti 2p . The signals for C, O, F, andTi can be detected in all samples, revealing the successful generationof abundant polar groups  on theMXene nanosheets during the etching process S8a.36 Th2, MXene–hemi4, MXene–hemi6, and MXene–hemi12 corresponds to 1.5%, 5%, 7%, and 10%, respectively. As shownin XRD patterns, pristine MXene film displays a peak at 6.8°(d-spacing of 13.0 Å) related to (002) planes(36 This peak shifted from 6.2° (d-spacing of 14.3 Å) to 5.5° (d-spacingof 16.1 Å) when the concentration of hemicellulose increasedfrom 2 to 12 mg mL–1 (Table S4). These results present a large amount of hemicellulose moleculesembedded into adjacent MXene nanosheets as a function of hemicellulosecontent, which increases the interlayer spacing.After verifying theoriented microstructures and molecular interactions,the electrical conductivity and mechanical properties of MXene filmswere further studied. ) planes3b.36 Thi2 to 125 MPa for MXene–hemi12, which is 6.6 times higher than that of the original MXenefilms (19 MPa). Similarly, Young’s modulus of MXene films alsoincreases with increasing hemicellulose content , which is consistent with the high conductivity of MXenefilm and MXene–hemi films.48 As for MXene–hemielectrodes, notably, the 45° angle section at the midfrequencyranges significantly reduces. In addition, the slope of the obliqueline at low frequencies slightly increases when compared to the pristineMXene electrode, indicating a lower ion transport resistance .51 In order to gain insightsinto the ion resistance, a complex capacitance analysis and frequencyresponse were conducted. The electrodes with hemicellulose show higherphase angle values at low frequencies and lower time constant (τ0), proving better ion response and kinetics , the dependence of the imaginarycapacitance on the real impedance was correlated .53 Among them, electrodeswith hemicellulose exhibit lower Ri values, further proving lower ion transport resistance. Hydrophilichemicellulose can act as a molecular spacer among MXene nanosheetswith increased interlayer spacing, which may facilitate ion transport.As a result, MXene–hemi electrodes possess a better rate performanceat high scan rates . Moreover, gravimetriccapacitances were measured of 366, 355, and 350 F g–1 for MXene–hemi2, MXene–hemi4, and MXene–hemi6 electrodes, respectively. A highcapacitance of 335 F g–1 was retained for the MXene–hemi12 electrode when compared to that of the pristine MXene electrodewith only 17% lower gravimetric capacitance (405 F g–1). However, the mechanical strength of the MXene–hemi12 electrode increased by 660% from 19 to 125 MPa. Therefore,the MXene–hemi electrode also shows decent integration of thehigh specific capacitance and mechanical strength, which is betterthan that for other reported MXene-based composite electrodes . The humidity-responseproperty of the MXene–hemi films was investigated through monitoringtheir electrical signal changes under various relative humidity values shows a peak at 6.2°  to 5.7° (d-spacing of 15.5 Å) when the relative humidity increased from35% to 95% (Table S5). These results revealthat a large amount of water molecules were uptaken into the adjacentMXene nanosheets as a function of relative humidity, resulting inan increase in the interlayer spacing and resistance of the film was obtained from Laizhou Kai Kai Ceramic Materials Co., Ltd. TheTi3C2Tx MXene dispersionin water solution was prepared according to a previous work.66 For detailed synthesis procedures, see the Surpporting Information.Tixylo-oligosaccharide) into the same volume of MXene dispersion. The concentration of hemicellulose was controlledat 2, 4, 6, and 12 mg/mL, respectively. The composite films were obtainedthrough simple vacuum filtration on a hydrophobic polyvinyl difluoride(PVDF) membrane with a pore size of 0.22 μm. The films weredried in a vacuum system and peeled off from the PVDF membrane. Theobtained films were named MXene–hemi2, MXene–hemi4, MXene–hemi6, and MXene–hemi12, respectively. For comparison, pristine MXene film was preparedwithout hemicellulose under the same conditions.Hemicellulose(xylo-oligosaccharide), obtained from corncob through pretreatment,enzymatic hydrolysis, flocculation, and subsequent purification, waspurchased from Shanghai Yuanye Bio-Technology Co. MXene–hemicellulosefilms were prepared by adding different amounts of hemicellulose (


Anableps anableps is composed of duplicated corneas and pupils, as well as specialized retina regions associated with simultaneous aerial and aquatic vision. In a previous transcriptomic study of the A. anableps developing eye we identified expression of twenty non-visual and eleven visual opsin genes. Here, we surveyed the expression territories of three non-visual melanopsins genes , one teleost multiple tissue opsin (tmt1b) and two visual opsins (lws and rh2-1) in dorsal and ventral retinas. Our data showed that asymmetry of non-visual opsin expression is only established after birth. During embryonic development, while inside pregnant females, the expression of opn4×1, opn4×2, and tmt1b spans the whole retina. In juvenile fish (post birth), the expression of opn4×1, opn4×2, opn4m3, and tmt1b genes becomes restricted to the ventral retina, which receives aerial light. Raising juvenile fish in clear water instead of the murky waters found in its natural habitat is sufficient to change gene expression territories of opn4×1, opn4×2, opn4m3, tmt1b, and rh2-1, demonstrating that different lighting conditions can shift opsin expression and potentially contribute to changes in spectral sensitivity in the four eyed fish.The development of the vertebrate eye is a complex process orchestrated by several conserved transcriptional and signaling regulators. Aside from partial or complete loss, examples of exceptional modifications to this intricate organ are scarce. The unique eye of the four-eyed fish  Change in environmental light is an important driving force for the diversity of visual systems in vertebrates. Aquatic animals occupy a great range of visual environments and have evolved adaptations to increase sensitivity to the availability of light . EvolutiDialommus fuscus and Dialommus macrocephalus, also known as “Rockskipper,” these fish are terrestrial predators and have a vertically split cornea that allows them to remain upright with one cornea in and the other out of the water. In addition, Bathylychnops exilis, inhabits depths between 600 and 3,000 ft, and has an auxiliary globe in addition to the main eyeball directed toward the ocean floor , expression of opn4×1a, opn4×1b, and opn4m3 are detected in horizontal, bipolar and amacrine cells while a small subgroup of retinal ganglion cells expresses opn4×1 and opn4m3. The expression of opn4m3 was also detected in the zebrafish retinal pigment epithelial (RPE) , five melanopsin genes were observed: opn4×1a and opn4×1b, opn4m1a1, opn4m1a2, and opn4m2 (tmt) is expressed in the eyes, brain, liver, kidneys, and heart of fugu (Fugu rubripes) and zebrafish and in the brain and eyes of medaka (Oryzias latipes)  . In the d opn4m2 . The nonlatipes) . Previoulatipes) . While nlatipes) .Anableps anableps.Asymmetric gene expression of opsin genes is another way of modulating visual sensitivity, and this mechanism plays an important role in the diversification of visual pigments in fish, leading to changes in spectral sensitivity . In addiAnableps are livebearers; female fish give birth to fully developed progeny, similarly to mammals. Previous work from our group has shown that the establishment of asymmetric expression of visual opsins (rh2-1 and lws) occurs prior to birth and is independent of photic input, suggesting that it is genetically programmed  and Bragança, Pará, Brazil (0°53′27.6″S 46°39′21.9″W). Animals were euthanized using MS-222  followed by decapitation.Specimens of A. anableps juvenile (up to 15 cm long) and larvae were collected and flash frozen in Tissue-Tek® O.C.T TM embedding medium . Sagittal cryosections (20 μm) of the eyes were obtained using CM1850 UV cryostat  and collected on ColorFrost Plus microscope slides , fixed in 3% paraformaldehyde  and stored at −80°C for further use.Eyes of wild-caught Eight juvenile fish were kept in a 75 Gallon glass aquarium with UV filter and standard fluorescent light (40 watts); fish were kept in dechlorinated tap water at 28°C with aeration and biological and mechanical filtration and fed daily with fish ration. Fish were exposed to a day and night cycle of 12 h for 30 days followed by euthanasia, according to Fuller and Claricoates .Sagittal sections (20 μm) were obtained using CM1850 UV cryostat , placed on ColorFrost Plus microscope slides  and stored at −80°C for further use. Eight wild-caught juvenile fish were used as control for these experiments.in situ hybridization for the genes opn4m3, opn4×1, opn4×2, and tmt1b, we adapted a PCR-based technique for the preparation of riboprobe templates  and dehydrated in 70, 95, and 100% RNase-free ethanol. Each section was then hybridized with a solution (32 μL) containing 50% formamide, 2× SSPE, 1 μg/μl BSA, 1 μg/μL poly(A)  in DEPC-treated water, and 1 μL of the DIG-labeled riboprobe. Slides were cover slipped, sealed by immersion in mineral oil, and incubated overnight at 65°C. Next, sections were rinsed in chloroform, de-cover slipped in 2× SSPE, and incubated serially in 2× SSPE/50% formamide, and in 0.1× SSPE at 65°C. Sections were then blocked for 30 min at RT in blocking buffer with 1% skim milk and incubated overnight with an alkaline phosphatase-conjugated anti-DIG antibody  Slides were washed twice for 15 min in TMN , and incubated in a detection solution containing the alkaline phosphatase substrates Nitro-Blue Tetrazolium Chloride (NBT) . and 5-Bromo-4-Chloro-3-Indolyl-phosphate p-Toluidine Salt (BCIP)  Slides were washed overnight, fixed in 4% paraformaldehyde (dissolved in PBS) and cover slipped with Cytoseal . Slides were imaged using a Nikon SMZ1500 microscope and processed on NIS-Elements D 4.10.01 program .protocol . Cryosecin situ hybridization was performed to examine the expression domains of three types of melanopsin subfamily of genes, opn4×1, opn4×2, opn4m3, and one type of teleost multiple tissue opsin, tmt1b, in the developing retina of pre-birth larvae. Our results showed that opn4×1, opn4×2, and tmt1b are expressed uniformly in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) in both dorsal and ventral regions of the retina  shifted and became uniformly expressed in the entire retina, while the expression of lws remained unchanged and restricted to the photoreceptor cells in the dorsal retina. While in captivity, animals were maintained in tanks with average temperature of 28°C, which closely mirrors that of their natural environment. The commercial diet provided for the animals in captivity, however, differs from their diet in natural settings. In the wild, the stomach content of A. anableps ranges from micro and macroalgae, insects, crustaceans, molluscs, fishes, to mud and detritus .in situ hybridizations (ISH) of A. anableps larvae. RR and JS designed the riboprobes for ISH. DS and LP performed light-induced experiments and analysis. DS, LP, and PS wrote the manuscript with input from BM, MA, JL, RR, and JS. All authors contributed to the article and approved the submitted version.DS, MA, JL, LP, and PS designed the research. LP, MA, JL, and BM performed


Objective: To explore the active components and epigenetic regulation mechanism underlying the anti-inflammatory effects of Lonicerae Japonicae Flos and Forsythiae Fructus herb-pair (LFP) in carbon tetrachloride (CCl4)-induced rat liver fibrosis.Methods: The main active ingredients and disease-related gene targets of LFP were determined using TCMSP and UniProt, and liver fibrosis disease targets were screened in the GeneCards database. A network was constructed with Cytoscape 3.8.0 and the STRING database, and potential protein functions were analyzed using bioinformatics analysis. Based on these analyses, we determined the main active ingredients of LFP and evaluated their effects in a CCl4-induced rat liver fibrosis model. Serum biochemical indices were measured using commercial kits, hepatocyte tissue damage and collagen deposition were evaluated by histopathological studies, and myofibroblast activation and inflammation were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. High-performance liquid chromatography-mass spectrometry was performed to determine the levels of homocysteine, reduced glutathione, and oxidized glutathione, which are involved in inflammation and oxidative stress.Results: The main active components of LFP were quercetin, kaempferol, and luteolin, and its main targets were α-smooth muscle actin, cyclooxygenase-2, formyl-peptide receptor-2, prostaglandin-endoperoxide synthase 1, nuclear receptor coactivator-2, interleukinβ, tumor necrosis factor α, CXC motif chemokine ligand 14, and transforming growth factor β1. A combination of quercetin, kaempferol, and luteolin alleviated the symptoms of liver fibrosis.Conclusion: The results of this study support the role of LFP in the treatment of liver fibrosis, and reveal that LFP reduces collagen formation, inflammation, and oxidative stress. This study suggests a potential mechanism of action of LFP in the treatment of liver fibrosis. Liver fibrosis is a complex compensatory repair response caused by chronic liver damage due to a viral infection, autoimmune disorder, or drugs, and is characterized by excessive deposition of extracellular matrix, ultimately leading to cirrhosis with high morbidity and mortality .The epigenetic regulation mechanism of liver fibrosis is intricate, mainly including DNA methylation, histone modification, microRNAs (miRNAs), etc., It regulates the occurrence and development of liver fibrosis by affecting the expression of fibrosis-related genes, the activation, proliferation, apoptosis of hepatic stellate cells (HSC) and myofibroblast (MF) differentiation. There is research  has showLonicerae Japonicae Flos, a dried flower bud or flower of the honeysuckle family, exhibits heat-clearing and detoxifying effects. Lonicerae Japonicae Flos has been reported to have a protective effect against liver damage induced by various factors  for the treatment of liver disease.  factors . Forsyth effects .The combination of the two herbs was first used in an ancient TCM, “YinQiao Powder,” which has detoxifying, heat-clearing, and anti-inflammatory properties. It was recorded in the famous works “Wen Bing Tiao Bian” and is prescribed for the treatment of hepatopathy. However, the mechanism of action of this combination in the treatment of liver fibrosis remains unclear.Due to the complex composition of TCM, its target pathways and mechanisms of action in the treatment of diseases are not clear; therefore, its scope of use is limited . NetworkLonicerae Japonicae Flos-Forsythiae Fructus herb-pair (LFP) using bioinformatics and constructed a component-target network. Additionally, we performed functional enrichment analyses of target genes to explore the possible therapeutic mechanisms in the treatment of liver fibrosis. We further used a carbon tetrachloride (CCl4)-induced rat liver fibrosis model to elucidate the mechanisms underlying the effects of LFP and its protective effects on the liver by investigating fibroblast differentiation, inflammatory reactions, energy balance, lipid metabolism, and oxidative stress.In this study, we combined network pharmacology and pharmacological experiments. First, we identified the main active components and targets of 4 was purchased from Macklin ; L-phenylalanine-d5  was purchased from CIL  and used as an analytical internal standard (IS); N-Ethylmaleimide , Tris (2-carboxyethyl) phosphine hydrochloride  was purchased from Sigma-Aldrich ; Anti-COX2 Antibody was purchased from BIOSS ; Anti-α-SMA Antibody from CST ; Anti-TGF-1β Antibody from SAB ; Goat anti-Mouse IgG (H + L) Secondary Antibody, Goat anti-Rabbit IgG (H + L) Secondary Antibody were purchased from CWBIO .Kaempferol, luteolin and quercetin were purchased from meilunbio ; Albumin (ALB), SOD and MDA kits were purchased from Nanjing Jiancheng (Nanjing Jiancheng Bioengineering Institute); Tumor necrosis factor-α (TNF-α) and interleukin-6 (Il-6) kits were purchased from Absin (Shanghai Absin Bioscience Inc.); CClMale SD rats (200–220 g) were purchased from the Guangzhou Ruige Biological Technology Co., Ltd.  2021-0059; the animal certification number was NO. 44827200000404). The rats were reared in a well-ventilated environment, with a temperature of about 20–25°C, a 12-hour light day and night cycle, free feed and drinking water, and were reared adaptively for 1 week. The experimental protocols were approved by the Animal Care and Use Committee of Guangzhou Medical University .https://old.tcmsp-e.com/tcmsp.php). The potential active compounds were screened based on their oral bioavailability and drug-likeness. The threshold was set as oral availability (OB) ≥ 30% and drug-like degree (DL) ≥ 0.18 because ingredients that meet these two criteria are considered potential active ingredients. The relevant target proteins of each active ingredient were screened in the TCMSP database, the gene name corresponding to the target was obtained using the Uniport (http://www.uniprot.org/), and the organism was selected as “human.”The chemical components and related targets of LFP were obtained by searching the pharmacological database and literature on traditional Chinese medicine systems . “Gifts≥48” was selected as the target for the treatment of liver fibrosis. After obtaining the target of the herb-pair on the active ingredient and the target of the disease, a Venn diagram was drawn by intersecting herb-pair active ingredient targets and disease targets based on “gene name.”“Liver fibrosis” was used as the key word, and the protein targets and corresponding gene names of liver fibrosis were obtained in the GeneCards (https://cn.string-db.org/) to construct a protein-protein interaction (PPI) network. High confidence (0.9) was used as the screening condition to filter out the isolated targets, limit the species to humans, and map the PPI network. Cytoscape3.8.0 built-in plug-in MCODE was used to perform module analysis on the PPI network, obtain potential protein function modules, and analyze and describe their functions.The common target information was imported into the STRING database  and Kyoto KEGG pathway enrichment analyses were performed.To further determine the biological processes and signaling pathways of the target protein genes, the targets of LFP in the treatment of liver fibrosis were entered into Metascape, the species was limited to humans, and LFP was first weighed in a conical flask, 100 ml of pure water was added, and incubated for approximately 30 min. The mixture was heated in a water bath at 99°C for 2 h, filtered, 100 ml of pure water was added again, and the mixture was heated in a water bath at 99°C for 1 h and filtered. The combined filtrates were concentrated to approximately 10 ml by evaporation. Methanol was added to the extract (ratio 1:10), the mixture was centrifuged at 15,000 rpm for 15 min, and the supernatant was collected. The chromatographic column used was Agilent ZORBAX SB-C18  using isocratic elution with a mobile phase consisting of 60% methanol and 0.1% formic acid. The flow rate was 1 ml/min, the column temperature was 40°C, and the detection wavelength was 360 nm.4-induced rat liver fibrosis group was administered blank solvent by gavage daily; 3) a positive control group was administered 50 mg/kg silybin by gavage daily; 4) a high-dose group (QLKH) was administered 102 mg/kg quercetin, 57 mg/kg luteolin, and 36 mg/kg kaempferol daily by the intragastrical route [i.g.]; 5) a moderate-dose group (QLKM) was administered 51 mg/kg quercetin, 28.5 mg/kg luteolin, and 18 mg/kg kaempferol daily by i.g.; 6) and a low-dose group (QLKL) was administered 25.5 mg/kg quercetin, 14.25 mg/kg luteolin, and 9 mg/kg kaempferol daily by i.g. For groups ii-vi, 0.6 mg/ml of 50% CCl4 (soybean oil: CCl4 = 1:1) were subcutaneously injected into the rat dorsum twice a week for 6 weeks; and for group i, 0.6 mg/ml of soybean oil were subcutaneously injected into the rat dorsum twice a week for 6 weeks , 36 rats were randomly divided into six groups: 1) a control group was administered blank solvent by gavage every day; 2) a CCl 6 weeks .4, the animals were fasted for 24 h, and their body weights were measured and recorded. The animals were then anesthetized with pentobarbital sodium (60 mg/kg) and euthanized. Blood samples were collected from the abdominal aorta for biochemical evaluation. Livers were immediately removed and weighed to calculate the relative liver index (liver index% = liver weight/body weight × 100%). Most of the liver was snap-frozen in liquid nitrogen, and the remaining tissue was fixed with 4% paraformaldehyde and processed.Forty-eight hours after the last administration of CClFixed livers were embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (H&E), and masson. Histological changes were evaluated by randomly selecting histology using an upright microscope .The levels of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (γ-GGT), total bilirubin (TBIL) were measured using a Chemray 240 Automatic Biochemical Analyzer . Albumin (ALB), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were evaluated using commercial kits, according to the manufacturer’s instructions. Tumor necrosis factor-α (TNF-α) and interleukin -6 (IL-6) was detected using commercialized ELISA kits according to the manufacturer’s instructions.−ΔΔCT method.Total RNA was extracted from the liver tissue using RNA extraction solution  according to the manufacturer’s instructions. The PCR primer sequences are listed in Proteins were extracted and quantified using the BCA method. The protein samples were subjected to electrophoresis, transferred to a membrane, blocked, and incubated with the following primary antibodies: anti-COX2 (1:1000), anti-TGF-1β (1:1000), anti-α-SMA (1:1000). The membranes were then washed thrice with 1 × tris-buffered saline, 0.1% Tween (TBST), incubated with horseradish peroxidase-labeled secondary antibody (1:3000) for 1 h, and washed thrice with TBST. After the ECL chemiluminescence reagent was added, membranes were scanned , and the images were analyzed using ImageJ software.2O solution at the appropriate concentration, and the reduction reaction was performed at room temperature. Next, 100 µl of NEM (50 mM) solution at the pertinent concentration was added to the first step of the reaction mixture, followed by the addition of 500 µl of cold methanol (50 ng/ml of Phe-d5), vortexing for 30 s, and incubation at −20°C for 60 min. The precipitated proteins were centrifuged  at 21,380 g for 15 min at 4°C, and the supernatant (600 µl) was collected and dried using a stream of dry N2 at 30°C. The residue was re-dissolved in 10 vol% aqueous methanol (100 µl), and the solution was vortexed for 1 min and centrifuged at 21,380 g for 15 min at 4°C. An 80 µl aliquot of the supernatant was collected for analysis.Serum (100 µl) was added to 20 µl TECP (100 mM) or ddH1/x), and GSSG =  × 307.33 × 2/612.63.Serum extracts were analyzed on a Sciex 4000 Triple Quadrupole HPLC-MS/MS system  using an ACQUITY UPLC BEH C18 analytical column . The mobile phase comprising 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) was supplied according to the following program: 0–1 min (5%–95% B); 1–2 min (95%–80% B); 2–3 min (80% B); 3–5 min (90%–80% B); 5–8 min (80%–5% B); and 8–10 min (5% B). The injection volume and flow rate were 10 μl and 0.3 ml/min, respectively. The multiple reaction-monitoring parameters are listed in t-test was used to compare the means between two datasets, and one-way analysis of variance (ANOVA) was used to compare the means between three or more data points. p < 0.05 was considered statistically significant.All experimental data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0.0 statistical software . The Student’s Lonicerae Japonicae Flos, corresponding to 449 targets, and 23 active ingredients of Forsythiae Fructus, corresponding to 510 targets were identified. After deleting the duplicates, a total of 31 active ingredients were obtained, and the combination had a total of four ingredients  and drug-like degree (DL). Under OB ≥ 30% and DL ≥ 0.18 as the screening conditions, 23 active ingredients of redients . After cIn the GeneCards database, using “liver fibrosis” as a keyword, 504 liver fibrosis disease targets with Gifts ≥48 were screened, and the target of the active ingredient of the herb-pair was intersected with the target of the disease. Finally, 93 targets related to honeysuckle-forsythia in the treatment of liver fibrosis were identified .The medicinal material-ingredient-target network contained 126 nodes and 450 edges. As shown in A PPI network containing 93 targets and 299 interaction lines was obtained using the STRING database to analyze the interactions of 93 drug-disease intersection targets and by setting high confidence (0.9) and hiding isolated targets . The PPIGO and KEGG analyses were performed using the Metascape platform. A total of 5676 GO items were obtained, including 395 for cellular composition, 645 for molecular function, and 4636 for biological processes. The enrichment results showed that the targets of the LFP were mainly distributed in the membrane raft, vesicle lumen, and transcription regulator complex , the maiThree active ingredients  of LFP were selected for further animal studies based on the previous database analysis and a literature search. To determine the ratio of the active ingredients in the drug combination, the standard calibration curves and linearity range of quercetin, luteolin, and kaempferol were investigated , and theThe concentrations of quercetin, luteolin, and kaempferol (QLK) in LFP were 16.84 μg/g, 9.36 μg/g, and 6.09 μg/g, respectively. The ratio of quercetin: luteolin: kaempferol was 34:19:12. Based on this ratio, we designed a dosing regimen for the subsequent animal experiments.4-induced rat liver fibrosis model group was administered blank solvent; 3) a positive control group that was administered 50 mg/kg silybin; 4) a high-dose group (QLKH) that was administered high doses of QLK; 5) a moderate-dose group (QLKM) that was administered moderate doses of QLK; 6) and a low-dose group (QLKL) that was administered low doses of QLK. For groups ii-vi, 0.6 mg/ml of 50% CCl4 was subcutaneously injected into the rat dorsum twice a week for 6 weeks; and for group i, 0.6 mg/ml of soybean oil was subcutaneously injected into the rat dorsum twice a week for 6 weeks.To evaluate the therapeutic effects of QLK, 36 rats were randomly divided into six groups: 1) a control group that was administered blank solvent; 2) a CClThe rats were then sacrificed, blood samples were collected from the abdominal aorta for biochemical evaluation, and the livers were immediately removed for histopathological analysis.p < 0.05) compared to that of the model group , whereas serum ALT, AST, TBIL, and γ-GGT levels, which were used to evaluate hepatocellular necrosis and cholestasis associated with hepatic fibrosis, were drastically increased compared to the control group. Interestingly, QLK treatment reduced serum ALT, AST, TBIL, and γ-GGT levels in a dose-dependent manner, alleviated CCl4-induced hepatic oxidative stress, increased serum SOD levels, and decreased MDA levels. QLK treatment also alleviated CCl4-induced hepatic inflammation and decreased TNF-α and IL-6 levels  and homocysteine (Hcy) levels reflect the degree of liver injury caused by inflammation and oxidative stress. Therefore, we detected the serum GSH and Hcy levels by LC/MS, and the results showed that the model group had higher GSH, GSSG and Hcy levels than the control group, and lower GSH/GSSG ratio. Notably, QLK treatment reduced GSH, GSSG and Hcy levels, and raised the GSH/GSSG ratio , indicatLonicerae Japonicae Flos and Forsythiae Fructus for the treatment of liver fibrosis. We provided a possible molecular mechanism of the protective effect of LFP and verified its targets and mechanisms using a CCl4-induced rat liver fibrosis model. The CCl4 has a wide range of hepatotoxicity and is in many ways similar to human chronic diseases. However, due to its high toxicity, it is necessary to pay attention to the concentration and administration time during operation. In this research, the designed dosage and frequency of administration can ensure a better incidence of liver fibrosis, and assure the survival rate of experiment animals.In this study, we combined network pharmacology and animal experiments to construct a “drug component-disease target” network by screening the main active components and targets of Lonicerae Japonicae Flos include flavonoids, iridoids, organic acids, and saponins, and the main chemical components of Forsythiae Fructus include alkaloids, flavonoids, phenethyl glycosides, triterpenes, and lignans  play a crucial role in the deposition of fibrotic tissues during liver fibrosis and cirrhosis . After lWe are concerned about that PRMT5 plays an important role in human cancers by promoting the cell proliferation, invasion, and migration, besides, PRMT5 inhibition was efficacious ameliorate leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPLW515L model of myelofibrosis. In addition, bromodomain-containing proteins (CPI-0610) combine with janus kinase inhibitor (ruxolitinib)for myelofibrosis that provide valuable ideas for the treatment of liver disease. We intend to design relevant research on the liver fibrosis model .4-induced liver fibrosis include PTGS2. PTGS2, also known as COX2, is an enzyme that is activated by various inflammatory factors, such as cytokines and bacteria. Under inflammatory stimuli, COX2 promotes inflammation and the synthesis of prostaglandins. COX2 has been also reported to play a crucial role in the occurrence and development of liver fibrosis  , plays aIn addition, methionine metabolism plays a pivotal role in liver fibrosis . Hcy is 4-induced liver damage (Lipid peroxidation is an important pathological mechanism underlying CClr damage . The liv4-induced liver fibrosis in rats. However, the mechanism of liver fibrosis alleviation by QLK needs to be further explored and verified in subsequent studies.In this study, we showed that QLK, a combination of two herbal TCM, alleviates the symptoms of CClIn summary, this study revealed that quercetin, luteolin, and kaempferol are the main active ingredients of LFP that mediate its effects in the treatment of liver fibrosis. QLK can activate FPR2; inhibit α-SMA, COX2, FPR2, PTGS1, NCOA2, IL-1β, TNF-α, CXCL14, and TGF-β1; and reduce collagen formation, fibroblast formation, inflammatory responses, and oxidative stress. Furthermore, LFP plays a hepatoprotective role by inhibiting hepatocyte apoptosis and ballooning degeneration. The results of this study may provide support for future research and clinical application on the role of QLK in human liver fibrosis.


Alkalihalobacillus oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using mass spectrometry-based secretome profiling, a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides was identified and termed CGTase-13. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C. Under the above conditions, the conversion rate of stevioside and rebaudioside A, two main components of steviol glycosides, reached 86.1% and 90.8%, respectively. To the best of our knowledge, this is the highest conversion rate reported to date. Compared with Toruzyme® 3.0 L, the commonly used commercial enzyme blends, glucosylated steviol glycosides produced using CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. Thus, CGTase provides a novel option for producing high-quality glucosylated steviol glycoside products and has great potential for industrial applications.The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Herein, the CGTase-producing strain  Stevia rebaudiana. Steviol glycosides have attracted considerable attention from researchers and engineers in the food industry because they are natural, non-toxic, etc. [Steviol glycosides are natural low-calorie sweeteners with high sweetness isolated from the leaves of ic, etc. ,2,3. Howic, etc. ,5,6.Stevia leaves; however, it exhibited a strong bitter aftertaste [Steviol glycosides are mixtures of diterpenoid glycosides, including stevioside (Stev), rebaudioside A (Reb A), rebaudioside D (Reb D), rebaudioside F (Reb F), rubusoside, and dulcoside A, and steviol aglycone is common in all the components ,8,9,10. tertaste ,14,15,16Cyclodextrin (CD) glucanotransferase  belongs to the α-amylase family 13 of glycoside hydrolases (GH13_2). With starch as the glycosyl donor, CGTase can transform steviol glycosides into glucosylated steviol glycosides ,21,22. T® 3.0 L from Novozymes is the most commonly used CGTase in the production of glucosylated steviol glycosides [® 3.0 L as a control. Herein, a CGTase-producing Alkalihalobacillus oshimensis strain was isolated from Stevia planting soil in Shandong Province, China. Using mass spectrometry (MS)-based secretome profiling, a CGTase was identified and termed CGTase-13. With soluble starch as the glycosyl donor, CGTase-13 transformed Stev and Reb A into glucosylated steviol glycosides with high efficiency. In addition, the glucosylated steviol glycoside products produced via CGTase-13 tasted better than those produced via Toruzyme® 3.0 L. The findings of this study will provide novel perspectives to establish a high-efficiency transglycosylation system for the catalysis of steviol glycosides into glucosylated steviol glycoside products with better taste.Presently, Toruzymeycosides ,23,24. TStevia planting soil in the Shandong Province, five strains exhibiting CGTase activity were further screened and cultured using Horikoshi medium plate. CGTases are unique enzymes that catalyze the conversion of starch to CD, which absorbs the dye contained in the Horikoshi media and forms a yellow halo zone. After assessing the transglycosylation ability of steviol glycosides via high-performance liquid chromatography (HPLC) analysis, an efficient CGTase-producing strain (wahahaMZJ-1) was selected for further studies. Based on physiological and biochemical characterization, 16s rDNA, and gyrB results, the isolated wahahaMZJ-1 strain producing CGTase was identified as A. oshimensis. This strain was preserved at the China General Microbiological Culture Collection Center with CGMCC 23164. The colony morphology is shown in Among the 191 strains isolated from A. oshimensis wahahaMZJ-1 catalyzed various glucosylated steviol glycosides. Among them, the contents of the mono- and di-(α1-4)-glucosylated products were higher. Furthermore, compared with the products produced via Toruzyme® 3.0 L, the products produced were different.HPLC was used to detect the glucosylated steviol glycoside product . The extBecause Reb D, Reb M, and their isomers are recognized as the components with better taste ,25,26,27A. oshimensis were significantly higher than those of the products catalyzed by Novozyme® 3.0 L.As shown in A. oshimensis extracellular enzyme exhibited higher contents of Reb D, Reb M, and their isomers than the products catalyzed by Toruzyme® 3.0 L. Therefore, it is speculated that a novel CGTase can be obtained from the extracellular enzyme for steviol glycoside modification, and the taste of the product is expected to be better than that produced via Toruzyme® 3.0 L.There is a phenomenon of “glucosyl group loss from mother ion ” in the process of MS . Despite this, the products catalyzed by The 12 possible CGTases were detected via liquid chromatography-tandem MS (LC–tandem MS/MS), as shown in Escherichia coli. Soluble starch (20 g/L) and steviol glycosides (20 g/L) dissolved in sterile water were incubated with 12 purified enzymes (20 mg/L) at 40 °C, and 220 rpm for 24 h and transglycosylation activity was detected. Among them, five CGTases exhibited relatively good transglycosylation activity  was incubated with the enzyme sample (20 mg/L) at 40 °C and 220 rpm for 18 h. The reaction product was diluted 10 times, and the concentrations of α-, β-, and γ-CDs were determined. The results revealed that the concentration of α-, β-, and γ-CDs were 0.152 ± 0.007, 0.322 ± 0.024, and 0.056 ± 0.005 mg/mL, respectively; hence, CGTase-13 belongs to β-CGTase.Soluble starch solution (20 g/L) prepared in 50 mM NaIt is easy to see that the optimum temperature was 40 °C a. For StFor Stev, the conversion rate increased with the extension of reaction time within 0–6 h and did not change much after 6 h. For Reb A, the conversion rate increased rapidly with an increase in time from 0 to 10 h and slowly with an increase in time from 10–18 h. After 18 h, the conversion rate remained almost unchanged. Therefore, 18 h was selected as the optimum incubation time c.Within the concentration range of 10–50 g/L, the conversion rates of Stev and Reb A gradually increase with an increase in soluble starch concentration. As the maximum solubility of the soluble starch used was ~50 g/L, it is impossible to continue to increase its concentration. Thus, 50 g/L was selected as the optimal concentration of soluble starch a.Within the concentration range of 4–8 g/L, with an increase in steviol glycosides concentration, the Stev conversion rate increased slightly, whereas that of Reb A decreased slightly. When the steviol glycoside concentration exceeded 8 g/L, the conversion rates of Stev and Reb A exhibited a downward trend. Considering that the concentration of steviol glycosides used in actual production is generally ≥10 g/L, and the conversion rate of Stev and Reb A decreases by ≤5% when 8 g/L is compared with 12 g/L, 10 g/L was selected as the optimal concentration of steviol glycosides b.Under optimum conditions, the conversion rates of Stev and Reb A reached 86.1% and 90.8%, respectively.® 3.0 L, the glucosylated steviol glycoside product produced by CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, stronger sweetness intensity, and higher overall preference. The overall preference score of the CGTase-13 catalytic product was 71.3, and that of the Toruzyme® 3.0 L product was 68.5. Thus, CGTase-13 improved the edulcorating quality of enzymatically modified steviol glycosides.The results of the sensory analysis are shown in ® 3.0 L. Therefore, it was speculated that there might be a novel efficient CGTase in the extracellular enzyme. Furthermore, there was a potential chance for improving the sensory profiles of steviol glycosides.Compared with steviol glycosides, an improvement in sweetness quality was observed in mono- and di-(α1-4)-glucosylated products. However, excessive glycosylation can affect the taste, such as decreasing sweetness and increasing bitterness ,23. NeveNumerous studies have employed CGTase alongside steviol glycosides as acceptor substrates to improve the gustatory characteristics of steviol glycosides ,23. The ® 3.0 L as a control, consistent with the literature [® 3.0 L, the glucosylated steviol glycoside product produced by CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. However, there remain some deficiencies in the aftertaste, such as a bitter taste, which warrant further improvement. This behavior resembles the results of Abelyan et al. [® 3.0 L. This indicates that CGTase-13 has obvious advantages compared with Toruzyme® 3.0 L, thereby providing a novel option for producing glucosylated steviol glycosides, and has great potential for industrial applications.To investigate the impact of modification, a sensory analysis was conducted using Toruzymeterature ,23,24. Cn et al. , who, den et al. ,30. ThusPrior studies have established that coupling and disproportionation activities are directly associated with transglycosylation activity . We seleStevia was planted were collected from the Shandong Province (China) and diluted in a sterile dilution solution . The aliquots were subsequently carefully positioned onto a Horikoshi medium plate and incubated for 3 days at either 28 °C or 37 °C. The isolates that produced CDs formed yellow halo zones. Once the isolates were definitively confirmed to secrete CGTases, the transformed products of steviol glycosides were analyzed via HPLC assays.Strain isolation and identification were adapted from Yu et al. , with mihttp://www.ncbi.nlm.nih.gov/genbank/index.html, accessed on 10 February 2021.).To identify each strain, each 16S rRNA gene sequence was amplified via polymerase chain reaction using the primer 27F/1492R (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′). The amplicons obtained were sequenced, and the 16S rRNA gene sequences were aligned with the sequences from GenBank  were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. A gel trypsin digestion method was performed to purify and prepare the gel piece (cut from an SDS-PAGE gel) before LC-MS/MS analysis.http://www.uniprot.org/, accessed on 7 May 2021.) using MaxQuant. Proteins were considered positively identified when they were identified with at least two tryptic peptides per protein. The false discovery rate was 1% for both peptides and proteins.The peptides were separated via EASY nLC and analyzed via MS using Q-Exactive . For LC analysis, separation was performed using a Thermo Scientific EASY column  pre-column, followed by a Thermo Scientific EASY column  at a flow rate of 300 nL/min. Full-scan MS spectra (m/z 300–1800) were acquired in the positive ion mode. MS files were searched against the Uniprot database (E. coli codon bias and further synthesized and cloned in pET-28a(+) via GENEWIZ . The cDNA sequences of CGTase-13 were also codon-optimized according to P. pastoris codon bias and further synthesized and cloned in pPIC9K via GENEWIZ. The sequence of CGTase-13 optimized for E. coli was submitted to GenBank and assigned the temporary accession number OP095271. The sequence of CGTase-13 optimized for P. pastoris was submitted to GenBank and assigned the temporary accession number OP095272.The cDNA sequences of CGTases were codon-optimized according to E. coli BL21 (DE3) competent cells for expression. To induce expression, 0.1 mM IPTG was added to the media. After incubation, the cells were harvested via centrifugation (4500× g for 10 min). The harvested cells were suspended in sodium phosphate buffer . The cells were lysed on an ice bath via sonication, followed by centrifugation at 48,400× g for 30 min at 4 °C to remove insoluble cell debris. The recombinant proteins were purified via fast protein LC on an ÄKTA purifier equipped with 5 mL HisTrap affinity columns . The columns were equilibrated with binding buffer  at a flow rate of 2.5 mL/min. A 30 mL sample was loaded into the first column and extensively washed with binding buffer for five column volumes. The columns were then eluted with elution buffer . The purified enzymes were collected, and the elution buffer was replaced with protein buffer .The verified plasmids harboring the desired genes were transformed into P. pastoris GS115 via lithium chloride transformation. The transformants were selected, cultured in 25 mL BMGY media, and incubated at 28 °C until the density at 600 nm (OD600) reached a value of 2–6. The transformants were centrifuged at 4000× g for 5 min to collect the cells, which were then inoculated into 100–200 mL buffered methanol complex media (BMMY). Then, 1% (v/v) methanol was added at 24 h intervals to induce the expression of the heterologous protein. After 4 days of induction, the cell cultures were centrifuged at 10,000× g for 5 min at 4 °C, and the supernatant was collected.The verified plasmid was digested using the restriction enzyme SacI and transformed into Strain isolation and identification were adapted from Yu et al. , with mioC/oC′ is the initial Stev/Reb A concentration, and tC/tC′ is the detected Stev/Reb A concentration after the reaction.The conversion rate was calculated as follows ,28:(1)StThe reaction mixture was monitored via HPLC . The chromatographic column used for screening the strains was SB-C18 . Other transglycosylation reactions used Hypersil NH2 column  for detection.The UPLC-ESI-MS analysis was conducted using Waters Acquity UPLC-Xevo TQ MS. The UPLC conditions are presented in The MS parameters were polarity: ES-, full-scan mode, capillary: 3.5 kV, cone: 19 V, source temperature: 150 °C, desolvation temperature: 550 °C, cone gas flow: 60 L/h, and desolvation gas flow: 700 L/h.2HPO4/NaH2PO4 (pH 6.0) was incubated with the enzyme sample (20 mg/L) at 40 °C and 220 rpm for 18 h. The reaction was quenched via incubation in a boiling water bath for 10 min. The reaction product was diluted 10 times, filtered through a 0.22-μm membrane, and analyzed using an evaporative light-scattering detector Alltech 3300 .A 20 g/L soluble starch solution prepared in 50 mM NaThe concentrations of α-, β-, and γ-CDs in the final sample were determined via UPLC using an Acquity BEH phenyl column  and eluted with a methanol/water ratio of 1:99 at 0.3 mL/min.2HPO4/NaH2PO4 (pH 6.0) were incubated with the enzyme sample (20 mg/L) at different temperatures  and 220 rpm for 24 h. The conversion rate was measured and calculated as described above.Soluble starch (20 g/L) and steviol glycosides (20 g/L) dissolved in 50 mM Na2HPO4/NaH2PO4 (pH 6–8), and 50 mM Tris-HCl (pH 8–9.8). Soluble starch (20 g/L) and steviol glycosides (20 g/L) dissolved in a different pH buffer were incubated with the enzyme sample (20 mg/L) at 40 °C, 220 rpm for 24 h. The conversion rate was measured and calculated as described above.The following buffers were used to evaluate the effect of pH on the conversion rate: 50 mM sodium acetate/acetic acid (pH 4–6), 50 mM NaSoluble starch (20 g/L) and steviol glycosides (20 g/L) dissolved in sterile water were incubated with the enzyme sample (20 mg/L) at 40 °C and 220 rpm for 0–24 h. The conversion rate was measured and calculated as described above.Soluble starch  and steviol glycosides (10 g/L) dissolved in sterile water were incubated with the enzyme sample (20 mg/L) at 40 °C and 220 rpm for 18 h. The conversion rate was measured and calculated as described above.Soluble starch (20 g/L) and steviol glycosides  dissolved in sterile water were incubated with the enzyme sample (20 mg/L) at 40 °C and 220 rpm for 18 h. The conversion rate was measured and calculated as described above.Sensory analysis was conducted by Zhucheng Haotian Pharmaceutical Co., Ltd., Zhucheng, Shandong. The experiments for sensory analysis were performed in a test room at a temperature of <30 °C. Eleven supertasters were recruited and requested not to eat or drink during the hour preceding testing, not to eat spicy foods during the previous day before each test, and not to use perfume on the day of testing. All stimuli were tested using a mouth sip-and-spit procedure. Each participant was provided with a cup of water. Participants were instructed to rinse their mouths between each sample and wait at least 2 min before continuing.®, 3.0 L, 500 ppm). Blind samples were provided to all the panelists for the sensory evaluation.The evaluated samples were provided as solutions and included enzymatically modified steviol glycosides  and enzymatically modified steviol glycosides (product transformed by ToruzymeThe method to assess the disproportionation activity of the CGTases was adapted with minor modifications from Kong et al. . The reaThe method to assess the coupling activity of the CGTases was adapted with minor modifications from van der Veen et al. . The reaThe method to assess the hydrolysis activity of the CGTases was adapted with minor modifications from Kong et al. . The reaThe method to assess the cyclization activity of the CGTases was adapted with minor modifications from previously described studies ,36 underAll activity data represents the mean value calculated from the results of three independent determinations.A. oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using MS-based secretome profiling, high-efficiency CGTase-13 was identified to convert steviol glycosides to glucosylated steviol glycosides. Under optimum conditions, the conversion rate of Stev and Reb A to glucosylated steviol glycosides catalyzed by CGTase-13 reached 86.1% and 90.8%, respectively. In addition, the sensory analysis revealed that the overall preference for glucosylated steviol glycosides catalyzed by CGTase-13 was higher than that for the product catalyzed by Toruzyme® 3.0 L. Thus, modified steviol glycosides catalyzed by CGTase-13 might be more suitable sweetener substitutes for artificial and caloric sweeteners in food formulations.The CGTase-producing strain


The development  was apparently affected in 3 dpf embryos. Furthermore, the alanine aminotransferase (ALT) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content in 6 dpf larvae were significantly increased. Additionally, the expression of heat shock response genes , inflammatory genes , and lipid synthetic genes  in 3 dpf embryos was significantly increased, which was also partially observed in the intestinal cell line form Pampus argenteus. Taken together, TFZ could affect the development of zebrafish, accompanied by disturbances of oxidative stress, heat shock response, inflammation, and lipid synthesis. Our findings provide an original insight into the potential risks of TFZ to the aquatic ecosystem.Triflumizole (TFZ) toxicity must be investigated in the aquatic environment to understand the potential risks to aquatic species. Accordingly, the adverse effects of TFZ exposure in zebrafish were investigated. Results demonstrate that, after TFZ exposure, the lethal concentration 50 (LC It is mostly used to prevent powdery mildew and rust in cereals, vegetables, fruit trees, and other crops [14-demethylation in sterol [The widespread use of pesticides may endanger aquatic species by contaminating water through surface runoff or leachingOerke ,2,3. Trier crops ,5,6,7. In sterol . As recen sterol . Furthern sterol . Environn sterol .50) of TFZ for rare minnow (Gobiocypris rarus) embryos was 7.11 mg/L, and that it induced abnormal development, extensively modified enzyme activities, and genes expression [Several studies have demonstrated the toxicity of TFZ to aquatic species, such as freshwater algae  and fishpression . TherefoMany aquatic species can survive in polluted environments because of defense mechanisms such as antioxidant system and stress response . In fishSome studies have reported that triazoles may affect lipid metabolism, harm embryonic development, and alter the expression of lipid synthetic genes ,22,23. IZebrafish have been commonly used to assess the toxicity of pesticides in the environment ,25. Our 2 anhydrous, 2.18 g; KH2PO4, 0.41 g; Na2HPO4 anhydrous, 0.142 g; MgSO4-7H2O, 4.9 g) to the exposure concentrations ensuring that the final DMSO volume was less than 0.1%.Triflumizole  was dissolved in dimethyl sulfoxide (DMSO) to 40 mg/mL as a stock solution, then diluted in 1 × EM medium   were maintained in a light/dark cycle of 14/10 h. The zebrafish were fed with freshly hatched brine shrimp twice daily. Water quality was monitored every day to ensure pH 7.3–7.4, conductivity within 450–500 μS, and a temperature of 28 ± 1 °C. Male and female (2:2) zebrafish were placed in isolation in the mating tanks in order to trigger spawning when the next morning lights on. After two hours, the normal embryos were collected and cultivated in 1 × EM medium at 28 °C.All zebrafish research procedures were approved by the Ethical Committee of Ningbo University. The AB strain adult zebrafish (An overview of the exposure procedures is shown in Pampus argenteus), was obtained and cultured as our previous method [The intestinal cell line obtained from a marine fish, silver pomfret , they were fixed on the culture dish in a lateral view using 3% methylcellulose. A lateral view of the entire zebrafish larvae was observed and photographed with a stereomicroscope .2O2 in 1 min at 25 °C and was expressed as μmol/min/g protein. The products of lipid peroxidation (measured MDA content) were tested by the thiobarbituric acid (TBA) method, and the amount of TBA substance that occurred through lipid peroxidation was detected after incubation at 95 °C with TBA. The pink color generated in these reactions was detected by spectrophotometry at 532 nm, and MDA content was expressed as nmol/g protein. The pyruvate reacts with 2-4-dinitrophenylhydrazine to generate the hydrazone, which acquires maximum staining by the addition of NaOH to measure ALT activity [2+ following protein-mediated reduction of Cu2+ by an alkaline environment. All measurements were performed on a microplate reader, using the A590 microwell plate protocol.Thirty embryos/larvae (each replicate) were homogenized with 300 μL saline solution, followed by centrifuging at 2500 rpm for 15 min at 4 °C to collect the supernatant for use in subsequent experiments. Three replicates were performed. The activities of SOD, CAT, alanine aminotransferase (ALT), and the content of MDA were assayed by commercially available biochemical assay kits  in accordance with the manufacturer’s instructions. The SOD activity was tested by the xanthine oxidase method. In brief, superoxide anions can oxidize hydroxylamine to nitrite and then turn amaranth purple in the presence of a chromogenic agent. Data were recorded by reading the absorbance at 550 nm and computing the activity of the SOD. One unit of SOD activity (U) was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/g protein. The CAT activity was tested by the ammonium molybdate method. The decomposition of hydrogen peroxide is quickly terminated by adding ammonium molybdate. The rest of the hydrogen peroxide reacts with ammonium molybdate to form a pale-yellow complex compound, which was detected at 405 nm and used to compute the CAT activity. One unit of CAT activity was defined as the amount of enzyme required to consume 1 μmol Hactivity . It was β-actin as an endogenous control gene [–ΔΔCT method [The total RNA was extracted using Trizol reagent  according to the manufacturer’s instructions. Next, the samples of RNA were reverse transcribed to generate cDNA using the Reverse Transcription Kit . Each 25 μL application contained 4 μL diluted cDNA, 10 μL qPCR PowerUp SYBR Green Master Mix , 1 μL forward and reverse primers (10 μM), and 4 μL RNA-free water. Two tests were performed for each gene in each biological replicate. The qPCR condition was as follows: 94 °C for 30 s (1 cycle), 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s (45 cycles). The results were subjected to relative quantitative analysis using rol gene . The priT method .50 values were determined using GraphPad Prism software version 9.0 . The data were expressed as mean ± standard error of means (SEM). The results were demonstrated through three independent experiments. One-way analysis of variance (ANOVA) with Tukey’s multiple range test was used to analyze cell activity differences. Differences among two groups were analyzed by unpaired t-test with Welch’s correction. * p < 0.05 was considered significant, and ** p < 0.01 was considered highly significant.SPSS version 20.0  was used for statistical analysis. LC50 of 3 dpf zebrafish embryos is 4.872 mg/L  and 1 mg/L (p < 0.05) TFZ groups than the control group  in 3 dpf zebrafish embryos was markedly increased in 1 mg/L TFZ group relative to the control group , grp78, hsp90, and grp94 in 3 dpf zebrafish embryos was significantly increased in 2 and 3 mg/L TFZ groups  in 3 dpf zebrafish embryos was significantly decreased in 2 mg/L TFZ group compared to the control group , interleukin 1, beta (il-1β), and cyclooxygenase type 2 a (cox2a) in 3 dpf zebrafish embryos was significantly increased in 2 mg/L TFZ group  and ppar-γ in 3 dpf zebrafish embryos was markedly increased in 1 mg/L TFZ group relative to the control group , acetyl-coa carboxylase (acc), and ppar-γ in 3 dpf zebrafish embryos was significantly increased in 2 mg/L TFZ group  and 20 mg/L (p < 0.05) TFZ groups , 10 (p < 0.01), and 20 mg/L (p < 0.01) TFZ groups relative to the control group  embryos was 7.11 (6.69–7.51) mg/L [Oncorhynchus mykiss, the acute 96 h LC50 was 0.57 mg/L in Lewis’s study [From our results, it is clear that TFZ strongly affected the early development of zebrafish in a concentration-dependent manner. After TFZ exposure, the 48 h LC51) mg/L , which w’s study , Hermsen’s study , which wIn addition, we discovered obvious morphological changes induced by TFZ, including pericardial edema, yolk sac swelling, liver size reduction and liver color darkening in 3 dpf zebrafish embryos after exposure for 48 h. Zebrafish embryonic toxicity tests found that pericardial edema and yolk sac retention were extensively noted after triazoles exposure ,30,31,32hsp70, grp78, hsp90, and grp94) in 3 dpf zebrafish embryos sharply increased in the 2 mg/L TFZ group, indicating a protective effect against protein misfolding [hsp90 in the intestinal cell line was significantly increased in the 5 and 20 mg/L TFZ groups, which was in accordance with 3 dpf zebrafish embryos.The antioxidant defense systems were affected by oxidative stress following TFZ exposure. SOD and CAT are potent enzymes for defending against oxygen toxicity because of their inhibition of effects on oxyradical formation . In addisfolding . Furthertnfα after exposure [p65-nfκb, il-1β, and cox2a which could be infer that potential tissue damage was caused by TFZ. The expression of cox2 was markedly upregulated in the 10 and 20 mg/L TFZ groups in the intestinal cell line, which was in accordance with zebrafish larvae.Several studies in aquatic organisms have demonstrated the potential for environmental pollutants to disrupt inflammatory gene expression. Inflammatory genes expressed in zebrafish larvae could be increased by glyphosate exposure . Tricyclexposure . Tissue exposure . Our stusrebp1, fas, acc, and ppar-γ in 3 dpf zebrafish embryos was markedly increased after TFZ exposure, which was in accordance with the expression of srebp1 and ppar-γ in the intestinal cell line. In reverse, TFZ caused liver damage and lipid synthesis through oxidative stress, heat shock response, and inflammation. Further studies should focus on the mechanism that TFZ contributes to lipid synthesis.The increased oxidative stress, heat shock response, and inflammatory response are closely related to lipid synthesis in zebrafish ,41. In oppar-γ suggests that the PPAR signaling pathway might participate in the imbalance of lipid metabolism. These findings provide a new insight into the potential mechanisms underlying the lipid metabolism imbalance of TFZ in zebrafish.In summary, our results show TFZ-induced morphological changes  as well as death in zebrafish embryos. TFZ exposure could cause oxidative stress in zebrafish larvae. Moreover, the upregulation of