Based on band structures, the band gaps reduce and the photocatalytic activities were extremely enhanced due to the shortening of electron transfer distance through the Z-scheme mechanism. Also, the graphenic structure of g-SiC is another parameter that was effective in improving photocatalytic performance by increasing the electron transfer and decreasing the rate of electron–hole recombination. Furthermore, the π back-bonding of g-SiC with metal atoms increases the electron–hole separation to enhance the photocatalytic activity. Interestingly, g-SiC composites (g-SiC/AWO) showed much higher photocatalytic properties compared to graphene composites (gr/AWO) and can remove the tetracycline even at dark by producing the oxygenated radicals via adsorption of oxygen on the positive charge of Si atoms in siligraphene structure.We developed here the efficient photocatalysts for the removal of high concentrations of tetracycline under visible light by immobilizing the AWO  nanocrystals on the surface of siligraphene (g-SiC) nanosheets. The g-SiC/AWO composites was synthesized by magnesiothermic synthesis of g-SiC and sonochemical immobilization of tungstates. These new heterojunctions of g-SiC/tungstates show superior photocatalytic activities in the degradation of high concentrations of tetracycline and 97, 98, and 94% of tetracycline were removed by using low amounts of g-SiC/Ag Wastewaters treatments are very expensive, and traditional methods in addition to imposing heavy costs do not meet the relevant standards in many cases. Recently, with the introduction of new technologies, new solutions such as novel photocatalysts, membranes, and environmentally friendly adsorbents have been introduced for wastewater treatments6. Photocatalysts are an important class of nanomaterials used in the purification of heavily polluted water and are very effective when other purification methods are not useful or economical10.Today, with the progress of human civilization and population growth, we are facing a severe water shortage and reduction of water quality due to various pollutions17, the tungstates with the general formula of A2WOx (AWOs) are considered to be the promising candidates with great photocatalytic potentials due to the unique electronic structure, wide band gap, good chemical stability, and physicochemical properties28. However, the high recombination rate of photogenerated electron–hole pairs reduces the potential of these catalysts to remove the high concentration pollutants28. To overcome this limitation, these photocatalysts combined with other materials to reduce the recombination rate. Graphene and its derivations are wildly used as metal-free co-catalysts to enhance photocatalytic performance by promoting charge transfer and reducing the rate of electron–hole recombination31.Among the wide range of metal-containing photocatalysts32, metal-free semiconductors such as g-C3N4 and g-SiC are also considered to be the most promising material for the construction of composite heterojunctions39.Also, interface engineering such as the fabrication of heterojunctions is an effective solution to improve the photogenerated charge separation in photocatalysts. In addition to 2D metal-containing semiconductors such as MXenes42. The g-SiC has high thermal, mechanical, and chemical stability along with high surface area, low band gap, and high electron mobility which made it a superior catalyst for removing different organic pollutants at high concentrations47. The 2D layered structure of siligraphene made it superior candidate to form the well-defined composite heterojunctions. Siligraphene can prevent the aggregation of composites as well as improve the separation of photogenerated electron − hole pairs due to the superior electron mobility of siligraphene. Also, the doping of Si atoms in this structure gives extraordinary properties to this compound compared to graphene, which can make this material a very good candidate for making improved catalysts with graphenic structures. So far none has reported the potential of siligraphene coupled with tungstates (AWOs) for removal of organic pollutants in water.Siligraphenes (g-SiCs) are the class of graphenic materials in which a large number of carbon atoms have been replaced by silicon2WO4, Bi2WO6, and Na2WO4 with siligraphene (g-SiC) were synthesized for removal of high concentration of tetracycline under visible light. Siligraphene (g-SiC) was synthesized via a low-temperature method to maintain the porous structure and g-SiC/AWO composites were developed by immobilizing of tungstates on the surface of g-SiC. Siligraphene can decrease the band gap, increase the charge transfer, and decrease the rate of photogenerated electron–hole recombination.In this work, the new Z-scheme heterojunctions of Ag-1 which confirms the formation of a bond between silicon and carbon. The g-SiC/Ag2WO4 spectra shows the stretching vibration of the Si–C bond at 785 cm-1 which is shifted to a lower frequency compared to g-SiC (813 cm-1), indicating the stronger Si–C bond. The stretching vibrations of the W–O bond appeared at 823 cm-1 and the bands at 570–650 cm-1 are related to W–O–W and O–W–O asymmetric stretching vibrations of the WO42- anion48.Figure -1 in g-SiC/Bi2WO6 catalyst which is slightly shifted to a higher frequency compared to g-SiC which indicates the immobilization of Bi2WO6 on the surface of g-SiC. The absorption bands that appeared at 582 and 470 cm-1 are related to Bi–O vibrations, and the absorption bands in the region of 729 cm-1 is corresponds to W–O vibration49.The Si–C stretching vibration was observed at 816 cm-1 in g-SiC/Na2WO4 catalyst. The W–O vibration of WO4–2 anion is observed in the 810 cm-1 and the band at 472 cm-1 is related to W–O–W vibration50.The absorption band of Si–C appeared at 837 cm2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 compared to g-SiC. The Ag2WO4 diffraction peaks were observed at 2Ɵ of 16.65, 29.95, 30.95, 33.15, 44.55, 46.15, 53.7, 55.6, 56.75, 65.01, and 77.9° in g-SiC/Ag2WO4 which represent the mixture of α and β phases of Ag2WO4  and 48.Figure 2WO6 sample, the diffraction peaks observed at 2Ɵ of 28.35, 32.95, 47.20, 56.16, 58.77, 76.06, and 78.61° indicate the (131), (200), (202), (310), (226), (113), and (139) planes of the orthorhombic structure of Bi2WO6 (JCPDS #39-0256)49.In the g-SiC/Bi2WO4 sample, the diffraction peaks of sodium tungstate were observed at 16.95, 27.75, 32.65, 43.40, 48.95, 52.05, 57.05, 59.95, 64.75, 67.45, 74.35, and 78.30° which indicate the (111), (220), (311), (331), (422), (511), (440), (620), (533), (444), and (642) planes (JCPDS# 12-772)51.Figure 47. The high amounts of immobilized tungstates (AWO) on the surface of g-SiC have led to the observation of weak diffractions from 6H-SiC in these composites.In all samples, the weak diffraction peaks of the hexagonal 6H-SiC structure were observed which indicates the covering of g-SiC surface with tungstate particles. 6H-SiC is a hexagonal polytype of SiC with stacking sequence repeats every six bilayers along the c-axis direction. The diffraction peaks of the g-SiC were observed at 31.61, 35.58, 37.98, 60.08, and 71.76 representing the (101), (102), (103), (110), and (202) planes in the hexagonal structure of g-SiC (JCPDS#-29-1128)The lattice fringe data were recorded in Table 2WO4, Bi2WO6, and Na2WO4 were observed on the surface of g-SiC in g-SiC/Ag2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 catalysts, respectively.Figure 2 adsorption/desorption isotherms of g-SiC, g-SiC/Ag2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4, which shows the decrease of surface area after immobilization of AWO on the surface of g-SiC. These results can be justified by the filling of pores in g-SiC by AWO particles, which is shown in pore size distribution curves .Figure ely Fig. . Results53. As shown in Fig. 2WO4, Bi2WO6, and Na2WO4, which indicates that the immobilization of tungstates on the g-SiC surface decrease the recombination rate of photogenerated electron-holes. In g-SiC/AWO samples, photogenerated electrons could be transferred efficiently from tungstates to g-SiC due to the high charge carrier of the g-SiC sheets.Photoluminescence (PL) spectra of synthesized samples were shown in Fig. 54. As shown in Fig. The efficiency of photogenerated electron–hole separation of synthesized samples was further studied by the electrochemical impedance spectroscopy (EIS)2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 in comparison with Ag2WO4, Bi2WO6, and Na2WO4 and their graphene composites (gr/AWO) were reported in Fig. 2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 after 20 min, respectively , and 16, 25, and 18% TCL removal were observed after 100 min  were also compared with g-SiC/AWO composites  is related to the surface charges in the g-SiC structure. According to our previous report, due to the electronegativity difference between Si and C in the g-SiC (siligraphene) structure, Si atoms have a partial positive charge and C atoms have a partial negative chargeAs shown in Fig. To achieve the best results, the reaction conditions were optimized in Fig. 2WO6 as the best photocatalyst , H2TC0 as the main species of TCL can be adsorbed on the surface of catalysts via interaction of negative tricarbonyl amide groups of TCL with a positive charge of Si atoms on the g-SiC structure10. At a pH lower than 3.3, the TCL removal decreases due to the repulsion between the H2TC+ species of TCL and the positive surface of catalysts. Also, at a pH higher than 7.7, the photocatalytic performances were decreased which is related to electrostatic repulsion between the negative surface of catalysts and HTC−/TC2− species of TCL  and conduction (CB) bonds. The CB and VB potentials of g-SiC/AWO catalysts were predicted by the following equation:Figure X is the electronegativity of the semiconductor and Ee is the energy of free electrons in the hydrogen scale (approximately 4.5 eV). The X value of g-SiC/Ag2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 are reported 6, 6.36, and 6.5 eV, respectively58. The Eg of Ag2WO4, Bi2WO6, and Na2WO4 were measured from UV–Vis spectra 3.1, 2.89, and 3.7 eV, respectively  is more negative than O2/·O2− (− 0.33 V) and the VB of Ag2WO4 (3.05 V), Bi2WO6 (3.3 V), and Na2WO4 (3.85 V) are more positive than OH-/·OH (+ 1.99 V), so, the photogenerated e− can reduce the O2 to form ·O2− radicals and photogenerated h+ can oxidize OH- to produce ·OH radicals. Results confirm the electrons in the CB of AWO combine directly with the holes in the VB of g-SiC, indicating the direct Z-scheme mechanism in the charge transfer path of photogenerated electron–hole in g-SiC/AWO heterojunctions.tra Fig. . As a re61. In this novel Z-scheme heterojunction not only gives photocatalyst with superior redox ability, but also accelerates the migration of photogenerated electron-holes62. Figure 2WO6 is the reason for the better photocatalytic activity of this catalyst.Traditional BWO-based heterojunction is commonly performed via the Type-II strategy to accelerate the rate of charge transfer. These conventional heterojunction has a drawback that charge migration causes the photogenerated electron–hole to have weak redox ability·OH and ·O2− radicals as the main species in the degradation of TCL. While, in g-SiC/AWO photocatalysts, ·OH and h+ are the reactive species in the photocatalytic reaction, and ·O2− radical shows the lower involvement in the degradation of TCL  can be developed to find out the visible light degradation of high concentration of antibiotics by improving the charge separation, enhancing the electron transfer in Z-scheme heterojunction, and decreasing the rate of photogenerated electron–hole recombination.2: 98%, porosity: 85%) was used as natural precursor of Si source and citric acid as a C source were purchased from Merck. Ethanol (96%), Mg powder (0.3–0.06 μm), hydrochloric acid (37%), and hydrofluoric acid (40%) were obtained from Merck.Perlite .FT-IR spectra were obtained from Bruker, Vector instrument. Scanning electron microscopy (SEM) was used to study the morphology of samples by TESCAN, VEGA3 microscope armed with the X-ray energy dispersive spectroscope (EDX), and TEM microscopy was done using Zeiss, EM10C microscope. Crystalline phases were identified by BrukerAxs, D8 Advance X-ray diffractometer (XRD) using CuK2 atmosphere to obtain the SiO2/carbon composite. To form the Si–C bond, the composite was mixed with Mg powder with the Si:Mg ratio of 1:2 and heated at 800 °C for 5.5 h under an N2 atmosphere (5 °C /min). To eliminate the impurity of MgO, the produced g-SiC was immersed in HCl solution (2 M) for 48 h and then washed with distilled water to remove MgCl2. The residual silica was removed by immersing the produced g-SiC in 2 M HF solution. Afterward, the g-SiC (siligraphene) was filtered and dried in a vacuum oven at 60 °C for 24 h.Citric acid was used as a carbon source and prepared by dissolving 0.8 g of citric acid in 100 mL of distilled water. Perlite powder was first washed with HCl (1 M) solution to remove its impurities and dried at 70 °C for 8 h. Then, 2 g of perlite was added to 200 mL of citric acid (8 g/L) solution and sonicated for 15 min. The powder filtered and dried at oven. To better penetration of citric acid to perlite pores, this process repeated 5 times. Finally, the mixture was dried at 80 °C for 24 h. The obtained powder was put in a furnace at 800 °C (5 °C /min) for 1 h under an N2WO4 and Bi2WO6 on g-SiC (siligraphene), 0.2 g of synthesized g-SiC was sonicated for 10 min in a certain amount of distilled water. Silver or bismuth solution was prepared by dissolving 0.1 g of silver or bismuth nitrate in 25 mL of distilled water and stirring for 0.5 h. Then, Ag or Bi solution was added to g-SiC suspension and stirred for 0.5 h. Tungsten solution was also prepared by dissolving 0.1 g of sodium tungstate in 25 mL of distilled water for 0.5 h, then added dropwise to Ag/g-SiC or Bi/g-SiC mixture under sonication. The final mixtures were placed in an oven at 160 °C for 5 h. The solid powder was filtered and dried at 70 °C.For stabilization of Ag2WO4 was also synthesized by adding 0.1 g of sodium tungstate to g-SiC suspension under sonication. The mixture was heated at 160 °C for 5 h. The solid product was filtered and dried at 70 °C.The g-SiC/Na2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 photocatalysts was dispersed in 10 mL of tetracycline (TCL) solution under stirring. The solution saturates with oxygen by air bubbling into the system and irradiated by visible light. To optimize the photocatalytic reaction, different concentrations of TCL  were tested by using 0.01, 0.015, and 2 g of catalyst at pH of 2–12. Before irradiation, all samples were stirred at dark for 5 min, and then the photocatalytic process was performed. The concentration of dye in the solution was measured by UV–Vis spectroscopy after each 20 min. After each 10 min, the concentration of TCL was measured by UV–Vis spectroscopy at 357 nm. The TCL removal percent was determined using the following equation:C0 and Ct are the initial and equilibrium TCL concentrations.To investigate the photocatalytic performance, the removal of tetracycline (TCL) as a drug pollutant were evaluated. In a batch experiment, a certain amount of g-SiC/Ag·OH) scavenger, 1,4-benzoquinone (BQ) as superoxide radical anions (·O2−) scavenger, and ammonium oxalate (AO) as the scavenger of the hole (h+).Quenching experiments were done by using 2 Mm of isopropanol (IPA) as hydroxyl radical  were synthesized and their photocatalytic properties were investigated in the removal of tetracycline. Based on the photocatalytic degradation results, the photocatalytic potentials of tungstates catalysts were promoted by immobilizing on the surface of g-SiC. The graphenic structure of g-SiC can improve photocatalytic performance by increasing the electron transfer and decreasing the rate of electron–hole recombination. Also, the π back-bonding of g-SiC with metal atoms increases the electron–hole separation to enhance the photocatalytic activity. Furthermore, the heterojunctions created with g-SiC reduce the band gap and can enhance the photocatalytic activity by shortening the electron transfer distance through the Z-scheme mechanism. The optimized g-SiC/AWO composites exhibited high photocatalytic performances and 97, 98, and 94% of high concentrations tetracycline (50 ppm) were removed after 20 min by using only 10 mg of g-SiC/Ag2WO4, g-SiC/Bi2WO6, and g-SiC/Na2WO4 catalysts, respectively. The g-SiC/Bi2WO6 photocatalyst shows the highest photocatalytic activity due to the smaller band gap and shorter electron transfer path.The new Z-scheme heterojunctions of siligraphene (g-SiC) with different tungstates (AgSupplementary Information.


Introduction: Inflammation is a defensive response of the body and the pathological basis of many diseases. However, excessive inflammation and chronic inflammation impair the homeostasis of the organism. Arachidonic acid (AA) has a close relationship with inflammation and is the main mediator of the pro-inflammatory response. Based on the prodrug principle, the new pharmaceutical compound aspirin eugenol ester (AEE) was designed and synthesized. However, the effects of AEE on key enzymes, metabolites and inflammatory signaling pathways in the AA metabolic network have not been reported.Methods: In this study, the anti-inflammation effects of AEE were first investigated in mice and RAW264.7 cells in LPS induced inflammation model. Then, the changes of the key enzymes and AA metabolites were explored by RT-PCR and targeted metabolomics. Moreover, the regulatory effects on NF–kB and MAPKS signaling pathways were explored by Western Blotting.Results: Results indicated that AEE significantly reduced the number of leukocyte and increased the lymphocyte percentage. AEE decreased the expression levels of IL-1β, IL-6, IL-8 and TNF-α both in vivo and in vitro. In the liver of mice, AEE downregulated the levels of AA, prostaglandin D2 (PGD2) and upregulated 12- hydroxyeicosatetraenoic acid (12-HETE). However, the changes of PGE2, PGF2α, 6-keto-prostaglandin F1α (6-KETO-PGF1α), 9-hydroxy-octadecenoic acid (9- HODE), 13-HODE, 15-HETE, docosahexaenoic acid (DHA) and thromboxane B2 (TXB2) were not significant. Additionally, it was found that AEE decreased the relative mRNA expression levels of p65 and p38 and the ratio of p-p65/p65.Discussion: It was concluded that AEE might inhibit the LPS-induced inflammatory response through the regulation of AA metabolism. This study provides the theoretical foundation for the development of AEE as a medicinal anti-inflammatory drug. Inflammation is the most common pathological reaction, which fights infection by releasing proinflammatory cytokines and inflammatory mediators. . AlthougArachidonic acid (AA) is a key constituent of cell membrane lipids. Based on three enzymes, cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP450), AA can be converted into a variety of metabolites . ProstagAs we all know, aspirin (Asp), which is the classic drug in the history of medicine, exerts a very significant therapeutic effect on inflammation . However50 of oral administration of AEE in mice was 10.937 g/kg  and Eug   is a white and odorless crystalline solid . Through937 g/kg , which w, p. o.) , indicat, p. o.) . Moreove, p. o.) . Pharmac, p. o.) . PreviouPreliminary experiments were conducted in mice and rats to test the anti-inflammatory, analgesic, and antipyretic effects . Howeverβ-actin (3700s), p65 (8242s), p-p65 (3033s), p38 (8690s), p-p38 (4511s) were obtained from Cell Signaling Technology., Inc. . The isotope internal standards including TXB2-d4 (319030), PGE2-d9 (10581), PGF2α-d4 (316010), 6-keto-PGF1α-d4 (15210), PGD2-d4 (312010), 9-HODE-d4 (338410), 13-HODE-d4 (338610), 12-HETE-d8 (334570), 15-HETE-d8 (334720), DHA-d5 (10005057), AA-d8 (390010) were purchased from Cayman Chemical Co., .Mouse macrophages (RAW264.7 cells) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. AEE (99.5%) was obtained from Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. . Asp and Eug were supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. . Lipopolysaccharide, trypsin, and Cell Counting Kit-8 were from Beijing Solarbio Science & Technology Co., Ltd. . Foundation FBS and DMEM high glucose medium were purchased from MCE Co., Ltd. . ELISA kits including TNF-α (JL10484), IL-1β (JL18442), IL-6 (JL20268) and IL-8 (JL20271) were supplied by Shanghai Jiang Lai Biotechnology Co., Ltd. . RNA extraction kit, reverse transcription kit, fluorescence quantitative PCR kit were from Mei5 Biotech Co., Ltd. . n = 8): Control group, Model group, Asp (100 mg/kg) group, Eug (90 mg/kg) group, AEE low  group, AEE medium  group and AEE high  group. AEE, Asp and Eug were mixed thoroughly with 0.5% CMC-Na to prepare the suspension. LPS was dissolved in sterile saline. The mice in the control and model groups received equal volumes of saline, while the mice in the other groups were administrated with the corresponding drugs daily for 7 days. After 30 min of the last administration, the mice were intraperitoneally injected with LPS (1 mg/kg) to induce the inflammation, while the mice in the control group were intraperitoneally injected with saline.The animal experimental protocol for this study was approved by the Animal Ethics Committee of Hebei Agricultural University . Fifty-six Kunming mice aged 3 weeks (20–30 g) were provided by SPF Biotechnology Co., Ltd. . The mice were raised under sufficient lighting, good air conditioning and ventilation, 50% relative humidity and 22°C room temperature. After 1 week of adaptation from purchase, the mice were divided into seven groups . The contents of TNF-α, IL-1β, IL-6, and IL-8 were detected by the corresponding ELISA kit  according to the manufacturer’s instructions.g, 4°C, 10 min). Next, the supernatant (500 μL) was purified to extract AA metabolites by Oasis HLB 96-well plate  which was activated by 2 mL methanol and 2 mL water. Then, the plate was washed successively with 2 mL Washing solution A and Washing solution B, and then eluted with 1 mL methanol. The solvent was evaporated by nitrogen. Then, the samples were redissolved in 50 μL acetonitrile/water  with 0.1% FA, and centrifuged  to obtain the supernatant for UPLC-MS/MS measurements.The liver sample (50 mg) was slowly thawed at 4°C, BHT protein precipitant (500 μL) and 10 μL of internal standard (1 μg/mL) were added, and then homogenized and centrifuged . The system is equipped with an ACQUITY UPLC BEH C18 column . The mobile phase flow rate was 400 μL/min, the column temperature was 45°C and the sample volume was 4 μL. solvent A was water with 0.1% FA and solvent B was acetonitrile with 0.1% FA. The liquid phase gradients were as follows: 0–1 min, phase B was maintained at 30%; 1–7 min, phase B linearly increased from 30% to 80%; 7–9 min, phase B linearly increased from 80% to 90%; 9–11 min, phase B was held at 90%. To ensure the stability of the assay process, equal amounts of quality control (QC) samples were mixed, and their relative standard deviation (RSD%) was evaluated. The RSD% results for the QC samples can be seen in The Mass Spectrometry (MS) analysis was conducted using a 5500 QTRAP mass spectrometer  in negative ion mode. The specific parameters used were as follows: a source temperature of 450°C, Ion Source Gas1 (Gas1) set at 55 psi, Ion Source Gas2 (Gas2) set at 60 psi, curtain gas at 30 psi, and Ion spray voltage floating (ISVF) at −4500 V. To measure the ion pair, multiple reaction monitoring (MRM) mode was employed for detection. Signal to noise ratio (S/D) was used to determine the limit of detection  and the limit of quantification . The LOD of the AA metabolites were ranged from 0.25–1,000 ng/mL. The results of quantification and confirmation masses, retention time and standard curves were summarized in β-actin selected as the internal reference gene. The RT-PCR reaction conditions at the setting of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°Cfor 30 s. The relative amount of mRNA transcription was determined using the 2−△△CT method.According to the kit instructions, total RNA was extracted from the liver tissue, and the mRNA was reverse transcribed into cDNA. The cDNA was then utilized in RT-PCR to assess the relative transcription levels of COX-1, COX-2, 5-LOX, PLA2, and CYP450 genes. The PCR primers were provided in 2. Asepsis was performed throughout and cell status and growth were observed daily.RAW264.7 cells were culturing in DMEM high sugar medium  in an incubator at 37°C and 5% COCytotoxicity was assayed by the CCK-8 method according to the instructions. Inoculate 96-well plates with cell suspension (100 μL/well) and place the plates in an incubator for 24 h. Add AEE solution at concentrations  and incubate for 24 h. Subsequently, 10 μL of CCK8 solution was added to each well, followed by 2 h incubation in the incubator. Finally, the absorbance at 450 nm was measured using a microplate reader, and the cell survival rate was calculated.The content of TNF-α, IL-6, IL-8, and IL-1β was measured using the ELISA method. The cells were divided into the following groups, control group (Control): normal culture for 24 h; inflammation model group (Model): 1 μg/mL of LPS for 24 h; LPS + ASP: pre-protected cells by adding 150 μM of ASP to the culture medium for 4 h and 1 μg/mL of LPS for 24 h; LPS + Eug: pre-protected cells by adding 150 μM of Eug to the culture medium for 4 h and 1 μg/mL of LPS for 24 h. Eug for 4 h and 1 μg/mL of LPS for 24 h. LPS + AEE : pre-protection of cells by adding 75 μM, 150 μM and 300 μM of AEE to the culture medium for 4 h and 1 μg/mL of LPS for 24 h. The RAW264.7 cells in the logarithmic growth stage were seeded in 48-well plates, and the supernatant from each group of cells was collected after the completion of the culture. The ELISA kit instructions were followed for further analysis.β-actin as an internal reference gene. Finally, the corresponding RT-PCR kit was used for detection following the manufacturer’s instructions. The reaction conditions for RT-PCR were 95°C for 30 s, 95°C for 5 s, 60°C for 30 s and 40 cycles. The relative transcript amount of mRNA was calculated using the 2−ΔΔCt method.The cells were cultured in 24-well plates, and after 24 h of culture, total RNA was extracted and analyzed for p65 and p38 content by reverse transcription. PCR primers can be found in g for 10 min at 4°C and collected the cell supernatant. The protein concentration was determined by the BSA method and the protein concentration was calculated from the standard curve. The protein was denatured by adding protein loading buffer and boiling at 100°C for 10 min. Gel electrophoresis was carried out and the proteins were transferred to PVDF membrane. The membranes were washed for 5 min in TBST, 3 times, and closed at room temperature for 1 h. The primary antibodies for p65, p-p65, P38, p-p38, and β-actin proteins were incubated overnight at 4°C and then incubated with secondary antibodies for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit and detected using a BIO-RAD imaging system . ImageJ 18.0 software was used to analyze the protein bands for grey-scale values.The RAW264.7 cells were treated with media containing different concentrations of AEE (150 μM) for 24 h, followed by 24 h treatment by LPS (1 μg/mL). The lysate was centrifuged at 14,000 post hoc comparison. When p ≤ 0.05, the difference was statistically significant. The chromatographic peak areas and retention times were extracted using Multi quant software . The AA content in the sample was determined based on the standard curve.Statistical analysis was conducted using SPSS version 26.0 . The data were expressed in means ± SD. One-way ANOVA was used for statistical analysis, followed by Tukey’s for p < 0.05) (p < 0.01). In comparison with the model group, the WBC in Asp group decreased significantly (p < 0.01), while LYMPH percentage in Eug group significantly increased (p < 0.05). Additionally, the results of WBC in Eug, AEEL, AEEM and AEEH groups were significant decreased than that in the model group (p < 0.05). The results of WBC, RBC, LYMPH, Gran, MCH, RDW, and MPV among AEEL, AEEM and AEEH groups were not significant (p > 0.05).The results of the routine blood analysis showed a significant decrease in both leukocyte count (WBC) and granulocyte percentage (Gran) in the control group compared to the model group ( < 0.05) . Conversp < 0.01) (p < 0.01). As compared with the model group, the level of TNF-α in Eug and AEE groups were significantly lower (p < 0.05), and Asp treatment also extremely reduced the TNF-α level (p < 0.01). Treatment with AEEM significantly decreased the levels of IL-1β, IL-6, and IL-8 (p < 0.01). There were no significant differences in the results of IL-1β, IL-6, IL-8 and TNF-α among the AEEM, AEEH, Asp and Eug groups (p > 0.05).The levels of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α were significantly increased in the mice of the model group compared to the control group ( < 0.01) . Regardip < 0.01) (p < 0.01). Moreover, the expression of PLA2 was significantly decreased in the AEE group compared to the Eug group (p < 0.01). There were no significant differences observed among the different doses of AEE groups (p > 0.05).We first detected mRNA changes in the enzymes of the AA metabolic pathway. The results revealed a significant increase in the expression of CYP450, PLA2, COX-2, COX-1, and 5-LOX in the model group compared to the control group ( < 0.01) . AEE tre2 (p < 0.05), while the concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) was significantly increased (p < 0.01) in the AEEM group , 12-HETE, TXB2, PGF2α, PGD2, docosahexaenoic acid (DHA), 15-HETE, 9-HODE and 6-KETO-PGF1α between model and AEEM groups (p > 0.05). In Based on the findings from the analysis of inflammatory cytokines and AA enzymes, AEEM exhibited a pronounced anti-inflammatory effect. Consequently, four liver samples from the AEEM group were chosen for targeted metabolomics analysis using LC-MS/MS. The results demonstrated a significant decreased in the concentrations of AA and PGDEM group . There wp < 0.01) (p < 0.05 or p < 0.01). There was no significant difference in the AEE group compared to the Asp and Eug groups. (p > 0.05).Compared with control group, CCK-8 assay showed that AEE treatment had no inhibitory effect on cell count, and the cell viability of 5, 15, 45, 75, 100, 150, 300, and 350 μM treatment was above 93% . In the  < 0.01) . AEE (15p < 0.01) (p < 0.05). In regard to p38 level, no difference was found between the model and Asp groups, while Eug and AEE  treatments significantly decreased p38 levels in comparison with the model group. Compared to the Asp and Eug groups, there were no significant differences observed in the AEE three groups in the results of p65 and p38 (p > 0.05). Furthermore, no significant differences were found among the three doses of AEE groups (p > 0.05).First, we conducted RT-PCR to assess the mRNA expression of p65 and p38 in RAW264.7 cells. The results demonstrated a significantly lower mRNA expression levels of p65 and p38 in the control group compared to the model group ( < 0.01) . AEE (15p > 0.05). Notably, treatment with AEE significantly inhibited the phosphorylation ratio of p65 in comparison to the model group (p < 0.05). Similarly, AEE showed inhibition on the ratio of p38 phosphorylation, but the result was not statistically significant (p > 0.05).In order to further investigate the mechanism behind the anti-inflammatory effect of AEE, we conducted Western blot analysis to examine the expression levels of p65 and p38 proteins. In 2, TXB2, PGF2α, PGD2, DHA, 15-HETE, 9-HODE and 6-KETO-PGF1α were increased. Subsequently, excess AA may lead to an inflammatory response through the activation of NF-κB p56 and MAPK p38. The results revealed that the AEE decreased the levels of inflammatory factors including TNF-α, IL-8, IL-6, and IL-1β. Furthermore, AEE treatment led to a trend of reduction in the concentrations of AA and its metabolites such as TXB2, PGF2α, PGD2, DHA, 15-HETE, 9-HODE, and 6-KETO-PGF1α. As the main inflammatory mediator, the level of PGE2 was not reduced by AEE treatment. COX catalyzes the conversion of AA to PGG2 and PGH2 (2 is synthesized from PGH2 under the action of microsome PGE synthetase-1 (mPGES-1), mPGES-2 and cytoplasmic PGE synthetase (2. Additionally, AEE decreased the ratios of p-p65/p65 and p-p38/p38 (In this study, it was observed that the inflammation triggered by LPS substantially elevated the AA concentration. Additionally, the expression levels of 5-LOX, COX-1, COX-2, PLA2, and CYP450 were upregulated. Moreover, the concentrations of their metabolites such as PGEand PGH2 . Then, Pnthetase . In this-p38/p38 .et al. used LPS to induce an inflammatory in mice and confirmed that probucol suppressed the total leukocyte count in response to inflammation (The quantity and ratio of blood cells serve as indicators of inflammation and prognosis in numerous diseases, reflecting the health status of the body. As an important indicator of immune function and body condition, the WBC is involved in important functions such as the immune response to disease and endocrine function. Zucoloto ammation . In thisIn vivo, LPS predominantly binds to toll-like receptors located on the surface of macrophages, triggering downstream signaling pathways to stimulate the production of various inflammatory factors, chemokines, and reactive oxygen species (et al. used 1 μg/mL of LPS to induce inflammation and oxidative stress model in RAW264.7 cells (In many inflammatory diseases, inflammatory factors become abnormal, and are important markers for the diagnosis of various diseases. Therefore, the downregulation of inflammatory factor expression plays a crucial role in disease management and prevention . LPS is  species . Previou.7 cells . Similar.7 cells . In pres.7 cells . The levin vitro and in vivo (et al. demonstrated that Asp specifically binds to PLA2 and has inhibitory effect on it (et al. showed that long-term administration of Asp resulted in an increase in the number of liver mitochondria and the levels of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase, affecting the metabolic activity of CYP450 (The enzymes COX, LOX, and CYP450 are responsible for catalyzing the synthesis of AA eicosanoids, which play a crucial role in inflammatory diseases . Inhibit in vivo . Singh ect on it . Wen et f CYP450 . It was in vivo (2 and TXA2 contributed to the production of pro-inflammatory cytokines such as IL-4, IL-5, and LTB4 (2, and TXB2 were increased in the model group. Rossi et al. demonstrated that decreased levels of 6-KETO-PGF1α in LPS-induced RAW264.7 cells might inhibit PG biosynthesis by interfering with AA release. (et al. showed that Asp reduced the levels of the inflammatory cytokine TNFα and TXB2 in a model of LPS-induced lung inflammation (1α, PGD2, PGF2α, and TXB2. These findings indicate that AEE may inhibit the LPS-induced inflammatory response by modulating the AA metabolic pathway.As important inflammatory biomarkers, AA and its metabolites are widely involved in immune and inflammatory responses in vivo . The livin vivo . Accumulin vivo . The impin vivo . Previouand LTB4 . In thisrelease. . Hamid eammation . In thiset al. suggested that compounds related to Eug may possess antioxidant and anti-inflammatory effects through the inhibition of NF-κB activation (The NF-κB and MAPK pathways are known as downstream inflammatory pathways of AA metabolism . NF-κB ptivation . The restivation . In additivation . Notably2 and PGE2 in whole blood should be analyzed to reflect the effects of AEE on the activities of COX-1 and COX-2. The signaling pathway can be further analyzed in depth through the methods such as knockdown or overexpression assays. Thirdly, the difference on the action mechanism between Asp and AEE on inflammation should be explored in further studies.Additionally, it is crucial to notice that the current study has certain limitations. Firstly, the number of AA metabolites tested in the experiment was few. In previous studies, 19 metabolites were detected . HoweverIn summary, AEE significantly inhibited the LPS-induced inflammatory response by reducing the levels of inflammatory metabolites in the AA metabolic pathway. In addition, AEE caused a reduction in the expression of p65 and p38 within the NF-κB and MAPK signaling pathways. These findings establish an experimental foundation for the potential clinical use of AEE in diseases associated with inflammation.


This review aims to provide a brief overview of CEP and summarize its recent findings in quantitative analysis, pharmacokinetics, therapeutic potential, and mechanism in antiviral and anti-SARS-CoV-2 activity. Given its remarkable capacity against SARS-CoV-2 infection  The emergence of the corona virus disease 2019 (COVID-19) triggered an unprecedented challenge to public health and human life worldwide since December 2019. This illness is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and presents as a severe acute respiratory syndrome. According to the latest statistics from the World Health Organization (WHO), over 638.17 million confirmed cases of COVID-19 and more than 6.61 million death cases have been reported globally. The COVID-19 pandemic has become the world’s largest and deadliest infectious disease outbreak, posing a serious global public health challenge that needs to be urgently addressed.In addition to implementing a strict dynamic zero-COVID-19 policy, China prevented and treated SARS-CoV-2 mainly through vaccines , neutralThe angiotensin-converting enzyme 2 (ACE2) receptor of SARS-CoV-2, which is also the surface receptor of SARS-CoV-2 spike glycoprotein, is expressed in alveolar epithelial cells and promotes the virus to enter and infect host cells . The serTM , one of the 3CL protease inhibitors from Pfizer; LagevrioTM (molnupiravir capsule), one of the RNA polymerase inhibitors from Merck; and VekluryTM (remdesivir injection), one of the RNA polymerase inhibitors from Gilead, are some of the drugs used in treating COVID-19. The State Food and Drug Administration (SFDA) in China conditionally approved the registration application of Azvudine tablets, the original anti-AIDS medicine, to add the indication for the treatment of COVID-19 on 25 July 2022. This became the first innovative small-molecule oral drug for the treatment of COVID-19 in China. However, information about the safety and effectiveness of these four innovative drugs is still limited in clinical use.PaxlovidThe high transmission and exponential growth rate of SARS-CoV-2 variants, coupled with the slow process of developing innovative drugs, have highlighted the need to quickly reuse existing drugs. This has led to the strategy of “new use of old drugs,” such as cepharanthine (CEP), as potential treatments for COVID-19. CEP is a conventional drug that has been used in Japan since 1951 to treat many acute and chronic diseases.It was recently identified as the most effective drug against SARS-CoV-2-related pangolin coronavirus, a less pathogenic model for SARS-CoV-2, in a large drug screen of 2,406 clinically approved drugs . Tong etGiven this finding and the close homology between the genome sequences of SARS-CoV-2 and SARS-CoV-2-related pangolin coronavirus, CEP has been shown with significant potential for treating COVID-19. In order to expedite the research and development of anti-COVID-19 medication, this review aims to summarize and analyze the current findings in quantitative analysis and pharmacokinetics, as well as to discuss its therapeutic potential and mechanism in anti-COVID-19 treatment.Stephania (Menispermaceae) such as Stephania japonica Miers, Stephania delavayi Diels, Stephania cepharantha Hayata, Epigeal Stephania Root, and long Stephania herb. It was initially separated and purified in 1934 by a Japanese pharmacist and named after one of its key sources: Stephania cepharantha Hayata. Chemically, CEP belongs to the family of BBIQ cyclic alkaloids, which includes tetrandrine, dauricine, curine, trilobine, neferine, daphnoline, and berbamine. Its chemical name is 6′,12′-dimethoxy-2,2′-dimethyl-6,7-[methylenebis-(oxy)] oxyacanthan, and its molecular formula is C37H38N2O6. The molecular weight of CEP is 606.71, and its structural formula is shown in CEP is a naturally occurring bisbenzylisoquinoline (BBIQ) alkaloid primarily derived from plants of genus CEP has been used as a conventional drug in Japan since 1951 to treat many acute and chronic diseases, including leukopenia , alopeciCEP has been shown to have multiple molecular mechanisms, including stabilizing cell membrane fluidity , inhibitIn recent years, it has been reported that CEP shows significant bioactivity against a range of viruses, including HIV-1 , hepatitCEP has recently been identified as the most efficient drug against SARS-CoV-2-related pangolin coronavirus, a less pathogenic model for SARS-CoV-2, in a large drug screening of 2,406 clinically approved drugs. Tong et al. from Beijing University of Chemical Technology obtained the national invention patent of China for pangolin coronavirus xCoV and its application, as well as the application of drugs against the coronavirus infection in May 2022. The patent specification demonstrated that a low concentration of CEP (10 μmol/L) could significantly inhibit coronavirus infection at the cellular level, with the virus content decreasing by 15,393 times compared to the control group. These findings were first published online in the National Medical Journal of China . A brief50 and CC50 ratio was over 70   -B, indic μmol/L) .The NSP12–NSP7–NSP8 complex of SARS‐CoV or SARS‐CoV‐2, which was essential for viral replication and transcription, was generally regarded as a potential target for antiviral therapy . In JuneIn vitro enzyme activity experiments were conducted to test the inhibitory ability of CEP on the ATPase activity of purified recombinant SARS-CoV-2 Nsp13 protein. The IC50 value was 0.4 μmol/L, indicating its noteworthy inhibitory ability on ATPase activity , which is critical for viral replication and the most conserved non-structural protein within the coronavirus family, using homology modeling and molecular dynamics approaches to screen ∼970,000 chemical compounds . In vitractivity . The sigactivity -D.In January 2021, Tong et al. from Beijing University of Chemical Technology conducted RNA sequencing to reveal the response and antiviral activity of cells to viruses. The results further confirmed that CEP could reverse the majority of the dysfunctional genes and pathways in virus-infected cells by interfering with heat shock factor 1 (HSF1)-mediated heat shock response, endoplasmic reticulum (ER) stress/unfolded protein response, and hypoxia pathway manipulated with by the virus. This also provided evidence for CEP as a promising therapeutic drug -A Li et.in vitro  as pan-coronavirus entry inhibitors, of which CEP exhibited the highest efficacy. According to this study, CEP could usefully protect different cell lines  from infection by different coronaviruses  in vitro -E , CEP, and remdesivir) had an EC50 value of less than 1 μmol/L against OC43 virus , to various target proteins including the spike protein, the main protease of SARS-CoV-2, NPC1, and TPC2 in humans  protein, leading to lysosomal cholesterol accumulation and elevated intra-lysosomal pH, which resulted in a cellular phenocopy of NPC -H Lyu e. Therefo50 and IC90 values of 1.90 and 4.46 μmol/L, respectively  are only predictive and not definitive proof that CEP engages these viral enzymes. Further research is needed to confirm these findings. Another limitation is that several of the cited studies in 50 value of CEP in vitro. Therefore, it is difficult to determine the specificity of the antiviral action independent of CEP-induced toxicity. Future studies should include both IC50 and CC50 measurements to provide a more comprehensive understanding of the antiviral potential and safety profile of CEP.In July 2022, Tong et al. from Beijing University of Chemical Technology established a cellular model using the coronavirus GX_P2V infection of Vero E6 cells in July 2022 . They usPharmacokinetic studies have become increasingly important in drug discovery and development. Such studies help evaluate concentration–effect relationships, design novel drug delivery systems, and optimize drug dosing regimens, among other things . Therefor2 = 0.999) with an LLOQ of 0.1 ng/mL. The study involved 12 SD rats, randomized equally into an intravenous group  and an orally treated group .max), half-life (t1/2), area under the concentration/time curve (AUC0-t), mean residence time (MRT0-t), clearance rate (CLz), and apparent volume of distribution (Vz) were determined to be 153.7 ± 16.18 ng/mL, 6.76 ± 1.21 h, 717.81 ± 158.35 ng/mL·h, 7.04 ± 0.49 h, 1.431 ± 0.31 L/kg·h, and 13.79 ± 1.76 L/kg, respectively  via the tail lateral vein, the peak concentration , with the LLOQ at 0.5 ng/mL. They tested different conditions to improve the poor solubility of CEP in water and found that an acidic vehicle was the key factor for improving CEP dissolution. Solutions of CEP with concentrations of 2.0 and 30 mg/mL were prepared in pH 3.7 and pH 2.8 acidic saline regulated by acetic acid, respectively, in which CEP powder was fully dissolved by vortexing for 20 s (n = 4), pulmonary , and oral administration  groups at a single dose of 1, 1, and 10 mg/kg, respectively. After a single intravenous or pulmonary administration of 1 mg/kg, CEP reached its Cmax in the plasma directly at the first sampling time with mean Cmax values of 148.8 ± 60.1 ng/mL and 65.3 ± 16.1 ng/mL, t1/2 of 2.80 ± 0.42 h and 16.35 ± 1.67 h, and AUC0-t of 576.2 ± 114.1 ng/mL·h and 392.2 ± 43.7 ng/mL·h  and pulmonary  groups at a single dose of 1 mg/kg. After a single intravenous or pulmonary administration of 1 mg/kg, CEP reached its Cmax in the plasma instantly at the first sampling time with mean Cmax values of 148.82 ± 60.08 ng/mL and 65.27 ± 16.05 ng/mL, t1/2 of 19.02 ± 4.46 and 14.64 ± 2.05 h, AUC∞0- of 597.4 ± 133.4 ng/mL·h and 382.7 ± 46.7 ng/mL·h, and MRT0-t of 18.9 ± 2.4 h and 30.4 ± 5.8 h, respectively . Compared with 1/2 of CEP in pulmonary administration was over 5.8 times higher than that in intravenous administration), it is confusing in a way. Anyway, these results confirmed that the absolute bioavailability of CEP could be significantly improved through pulmonary administration via the trachea using a nebulizer and laryngoscope, which indicated that aerosol inhalation of CEP might be a potential method. However, the limitation was the minor sample size (n ≤ 4) of these two studies.for 20 s . Twelve  ng/mL·h -C, D . After a single oral gavage administration of 40 mg/kg of CEP-SEDDS or CEP suspension, CEP reached its Cmax values of 1.00 ± 0.48 μg/mL or 0.43 ± 0.06 μg/mL, separately, whereas the Tmax value was 2.92 ± 0.20 h or 4.25 ± 2.95 h, and t1/2 was 8.65 ± 4.97 h or 4.86 ± 3.08 h, respectively  and infected mice (n = 24) received intraperitoneal (i.p.) administration of CEP at a dose of 21 mg/kg. The mean values of Cmax, Tmax, t1/2, AUC0-t, CL/F, and Vd/F of CEP in healthy mice were 874 ng/mL, 0.25 h, 2.52 h, 2,069 ng/mL·h, 8.77 L/h·kg, and 31.8 L/kg, respectively  and found the corresponding values of Cmax, t1/2, AUC0-t, AUC0-∞, MRT0-t, CLz, and Vz to be 262.6 ± 5.09 ng/mL, 14.55 ± 2.46 h, 1,376.85 ± 428.45 ng/mL·h, 1,506.83 ± 434.82 ng/mL·h, 12.24 ± 1.98 h, 2.14 ± 0.66 L/h·kg, and 44.77 ± 5.76 L/kg, respectively (ectively -A (Desgrectively . Correspectively -B (Desgrectively . Accordinfection . In our o humans . As a coectively -C.n = 4) at a dosage of 200 mg/kg. The plasma concentration of CEP in whole blood decreased rapidly from 0.49 ± 0.02 μg/mL immediately after injection, and the decrease rate slowed down after 2.25 h, reaching equilibrium between 24 and 72 h. The concentration–time curve of CEP in rabbit blood was consistent with that of a two-compartment open model. The values for t1/2a and t1/2β were 0.204 h and 4.62 h, respectively , and pharmacokinetic parameters in this study were reported as means without corresponding standard deviations.Rabbits are commonly used as animal models in pharmacokinetic studies . Li et aectively . The Vd ectively -D. The rn = 2), 30 mg (n = 5), 60 mg (n = 5), and 120 mg (n = 5), intravenously injected (single treatment in 5 min) at the doses 25 mg (n = 5), 50 mg (n = 5), and 100 mg (n = 5), and continuous intravascular administration (single treatment in 5 min for each time) at doses of 100 mg/day (n = 5) for 7 days, respectively  and trough (Cmin) concentrations of CEP were 196.3 ± 26.50 ng/mL and 34.0 ± 2.32 ng/mL, respectively, and the t1/2 was 62.0 ± 2.8 h (1/2 was about twice as long as that observed after a single intravenous administration (r2 = 0.9994) with the LLOQ at 0.5 ng/mL  than that reported by Yasuda et al.  ( > 0.05) . Hence, Furthermore, CEP was frequently distributed in matured T cells in the thymus . Moreovein vitro using human liver microsomes (HLMs) with specific probe actions and probe substrates. They found that only the activity of CYP3A4, CYP2E1, and CYP2C9 was inhibited by CEP, with IC50 values of 16.29, 25.62, and 24.57 μmol/L, respectively. Enzyme kinetic studies showed that CEP was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2C9, with inhibition constant (Ki) values of 8.12, 11.78, and 13.06 μmol/L, respectively . It has been used as a safe, well-tolerated, and inexpensive drug since 1951 to treat many acute and chronic diseases, such as leukopenia , alopeci in vivo . Monothemaxin vivo/IC50in vitro to ensure whether the concentrations of the cellular effects of CEP are kept in perspective with the antiviral concentrations and what is actually achievable in humans. The IC50 values of CEP were different for various virus strains infecting different cell lines. The lowest IC50 value in the available data so far was 28.51 ng/mL (0.047 μmol/L), which occurred in the 293T-ACE2 cell line infected with SARS-CoV-2 N501Y.V1 (B.1.1.7). We used this IC50 (28.51 ng/mL) as a reference standard to calculate the ratio of Cmaxin vivo/IC50in vitro, as shown in It is necessary to calculate the ratio of Cmax of CEP can be achieved with i.v. administration, particularly the trough concentration and peak concentration of steady-state blood drug concentration after 7 consecutive days/IC50, which are 1.19 and 6.89, respectively, indicating that effective antiviral concentrations can be achieved in humans. However, due to the poor bioavailability of CEP in vivo, the ratio of Cmax/IC50 was only 0.24 when administering CEP at a single oral dose of 120 mg. This implies that further research is needed concerning dosage form modification or route of administration to increase the bioavailability of CEP. Additionally, by optimizing the structure of the lead compound CEP and changing its main metabolic pathway, the metabolic stability of the compound can be effectively improved, the action time of the drug in vivo can be prolonged, the exposure in vivo can be increased, the bioavailability can be improved, and the pharmacokinetic characteristics can be optimized.According to max of CEP could be achieved with i.v. administration, an i.v. treatment is not a therapy that can be globally distributed and accessible. However, unfortunately, when CEP is administered at a conventional oral dose, the Cmax of CEP in humans is so low that it is far from being close to the in vitro IC50 value for blocking SARS-CoV-2. Pharmacokinetic studies in animals confirmed that the absolute bioavailability of CEP obtained obviously improved (achieved over 64%) by pulmonary administration  to remove PAINS from screening libraries and exclude them from bioassays (in vitro.It is important to consider whether CEP is a pan-assay interference compound (PAINS), given its bioactivity in so many ew drugs . For exaew drugs . To avoiew drugs . They deioassays . This PAioassays . We havein vivo  undertaken by Xinhua Hospital affiliated to Shanghai Jiao Tong University School Of Medicine and Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. This is an interventional efficacy and safety, phase II, double-blind, 3-arm study to investigate orally administered high-/low-dose CEP compared with placebo in nonhospitalized asymptomatic or mildly symptomatic adult participants with COVID-19, and the results have not yet been published  for the enteric-coated formulation of CEP , which showed good absorbance and remarkably improved bioavailability, and submitted a phase I/II clinical trial application for PD-001 to the FDA to treat the SARS-CoV-2 infection and obtained an agreement on the development of PD-001 toward clinical studies for mild-to-moderate COVID-19 , specifiublished .max value of CEP, it is not a practical treatment option for global distribution and accessibility. However, when administered at a conventional oral dose, CEP Cmax in humans is too low to be effective against SARS-CoV-2 in vitro. Pharmacokinetic studies in animals showed that pulmonary administration significantly improved the absolute bioavailability of CEP, reaching over 64%  Miers, Stephania delavayi Diels, Stephania cepharantha Hayata, Epigeal stephania root, and long Stephania herb. These plants possess a mature technique for extraction with low prices, and CEP has the advantages of safety, effectiveness, and accessibility. In conclusion, considering its remarkable capacity against SARS-CoV-2 infection in vitro and in vivo, with the lungs as the fundamental target organ; its good pharmacokinetic profile; and its advantages of safety, effectiveness, and accessibility, CEP is a promising drug candidate for treating COVID-19 (As an approved drug that has been used for more than 70 years, CEP is mainly extracted, separated, and purified from plants of the COVID-19 .


High-entropy alloys (HEA) with superior biocompatibility, high pitting resistance, minimal debris accumulation, and reduced release of metallic ions into surrounding tissues are potential replacements for traditional metallic bio-implants. A novel equiatomic HEA based on biocompatible metals, CrMoNbTiZr, was consolidated by spark plasma sintering (SPS). The relative sintered density of the alloy was about 97% of the theoretical density, indicating the suitability of the SPS technique to produce relatively dense material. The microstructure of the sintered HEA consisted of a BCC matrix and Laves phase, corresponding to the prediction of the thermodynamic CALPHAD simulation. The HEA exhibited a global Vickers microhardness of 531.5 ± 99.7 HV, while the individual BCC and Laves phases had hardness values of 364.6 ± 99.4 and 641.8 ± 63.0 HV, respectively. Its ultimate compressive and compressive yield strengths were 1235.7 ± 42.8 MPa and 1110.8 ± 78.6 MPa, respectively. The elasticity modulus of 34.9 ± 2.9 GPa of the HEA alloy was well within the range of cortical bone and significantly lower than the values reported for commonly used biomaterials made from Ti-based and Cr–Co-based alloys. In addition, the alloy exhibited good resistance to bio-corrosion in PBS and Hanks solutions. The CrMoNbTiZr HEA exhibited an average COF of 0.43 ± 0.06, characterized mainly by abrasive and adhesive wear mechanisms. The CrMoNbTiZr alloy’s mechanical, bio-corrosion, and wear resistance properties developed in this study showed a good propensity for application as a biomaterial. The Ec 142 GPa) ,55, 316L142 GPa) , and sim142 GPa) . TherefoCrMoNbTiZr HEA fractured at approximately 45° to the compression axis, as shown in the macroscopic image in 2) was slightly lower than in Hanks’ (650 µA/cm2), indicating that passivation occurred faster in PBS. Complete surface passivation occurred at 907.25 and 906.83 mA/cm2, respectively, in PBS and Hanks solutions. At this point, a further increase in potential did not result in any measurable current density. Overall, the corrosion rate (Cr) in PBS (1.24 × 10−3 mm/yr) was lower than in Hanks’ solution (2.03 × 10−3 mm/yr).Potentiodynamic polarization curves of CrMoNbTiZr HEA in PBS and Hanks physiological solutions at room temperature are shown in ace film ,51. At t2) is lower than the values reported for some commonly used biomedical alloys, such as CoCrMo in PBS (18.57 µA/cm2) [2), and additively manufactured 316L in PBS (0.57 µA/cm2) [The primary passive potential (bTa HEAs  in HanksbTa HEAs . Neverth µA/cm2) , wrought µA/cm2) . A summa3+ + 3 e− ⇌ Cr(s), 3+ + 3 e− ⇌ Mo(s), 3+ + 3 e− ⇌ Nb(s), 3+ + 3 e− ⇌ Ti(s), 4+ + 4 e− ⇌ Zr(s), SEM images b,c of th−1.45 V) .−4 mm3/Nm, which was lower than the 4.0 × 10−3 mm3/Nm for CuMoTaWV [−4 mm3/Nm for a typical biomedical Ti6Al4V alloy [CuMoTaWV  and 3.5 4V alloy .0.5Mo0.5C) are also expected to enhance the wear resistance of the alloy, as observed in some earlier reports [This result indicates the high wear resistance of the HEA. The enhanced wear resistance in this study may be associated with the high microhardness of the BCC and Laves phases. Moreover, in situ reinforcing carbides of Nb (NbC) and Ti-Mo (Ti reports ,64. High reports  due to tThe totality of the experimental results and discussions indicate that microstructural design through the selection of the constituent elements greatly influenced the microhardness, compressive and wear properties of the alloy. It can be concluded that the mechanical, bio-corrosion, and wear-resistance properties of the CrMoNbTiZr HEA developed in this study showed a good propensity for application as a biomaterial.2 in PBS and Hanks’ solutions. These translated to low corrosion rates of 1.24 × 10−3 and 2.03 × 10−3 mm/yr in PBS and Hanks’ solutions, respectively. CrMoNbTiZr HEA exhibited an average COF of 0.43 ± 0.06, characterized mainly by abrasive and adhesive wear mechanisms. The totality of these results showed that microstructural design through the selection of the constituent elements greatly influenced the mechanical and electrochemical properties of the alloy. The HEA developed in this study showed a good propensity for application as a biomaterial due to good mechanical properties and excellent bio-corrosion resistance. Finally, further research is required to investigate the fretting wear behavior and assess other in vitro and in vivo cell proliferation properties of the alloy.A novel equiatomic CrMoNbTiZr HEA was synthesized by elemental powder mixing and consolidated by spark plasma sintering. The relative density of the alloy approached 97% of the theoretical density, indicating the suitability of the spark plasma sintering technique to produce relatively dense material. The microstructure, mechanical properties, and bio-corrosion behavior of the HEA were investigated. The microstructure consisted primarily of BCC and Laves phases, which matched the prediction from a thermodynamic CALPHAD simulation. During sintering, carbides of Nb and Mo were formed due to the diffusion of carbon from the graphite die. The HEA exhibited a global Vickers microhardness of 531.5 ± 99.7 HV, while the individual BCC and Laves phases had values of 364.7 ± 99.4 and 641.8 ± 63.0 HV, respectively. The ultimate compressive strength and compressive yield strength were 1235.7 ± 42.8 MPa and 1110.8 ± 78.6 MPa, respectively. Its elasticity modulus of 34.9 ± 3.1 GPa was significantly lower than the values reported for commonly used biomaterials made from Ti-based, Cr–Co-based, and stainless-steel alloys. Importantly, it was well within the range reported for cortical bone. The alloy exhibited good bio-corrosion resistance with low current densities of 0.14 and 0.24 µA/cm


Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease  They arClostridium histolyticum (accession MER0000831) and is a member of enzyme family C11. Peptidases in this family are characterized by the presence of a catalytic cysteine-histidine dyad with a preference for hydrolyzing arginine and lysine bonds in the P1 position (2+ and/or reducing agents such as dithiothreitol (DTT) for activation and catalysis. Variance in the number of cleavage site(s) for activation is also observed, and in some cases, an amino acid linker peptide is removed , located at the southern part of the Mohns Ridge , 2010 in−1 . In Junencubator  consistincubator , was dep2.2.For the primary metagenome, one plate of 454 GS FLX Titanium shotgun reads  was sequenced and assembled using the Newbler assembler v.2.8  with a minimum identity of 96% over a minimum of 35 bases. In total, 0.9 million (75%) of the 454 raw reads were assembled into contigs resulting in 7448 contigs >500 bp and an N50 contig size of 10,887 bp. Open reading frame (ORF) predictions were made using Prodigal v2.60  and scre2.3.Sequence alignment was made using the ESPript 3.0 utility . The amiThe translated globupain DNA sequence  was subm2.4.Escherichia coli expression (NdeI and XhoI) into pET-21A by GenScript, omitting the predicted 21 amino acid signal peptide  was synthesized by GenScript  and codon-optimized for pression . The gen2.5.E. coli cells  using a heat-pulse manual supplied by the manufacturer. Cells were spread onto LB-agar plates supplemented with 100 μg/mL ampicillin and incubated at 37°C overnight. Pre-cultures were inoculated by picking one single colony and incubating at 37°C in LB media containing 100 μg/mL ampicillin with 190 rpm shaking overnight . Expression cultures were inoculated with 5% of pre-culture in LB media with 100 μg/mL ampicillin at 37°C and 190 rpm. At OD600 of 0.6, the temperature was set to 20°C, and the culture was equilibrated for 30 min. Heterologous expression was induced by IPTG brought to 0.1 mM IPTG, followed by overnight incubation (20°C). Cells were harvested by centrifugation at 5,500 rpm for 15 min at 4°C . Pellets were stored at −20°C.Expression plasmid of globupain and substitution variants were transformed into BL21-Gold (DE3) chemically competent For purification of globupain and substitution variants, cells were resuspended in lysis buffer , placed on ice for 30 min, and lysed by ultra-sonication . The lysate was clarified by centrifugation at 5,500 rpm for 20 min at 4°C . The sample was then loaded into a HisTrap HP 5 mL column  equilibrated with 20 mM HEPES, 500 mM NaCl, 25 mM imidazole, pH 7.5 with a flow rate of 1 mL/min. After elution with 20 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.5. fractions with the highest amount of enzyme were pooled and concentrated. The buffer was changed  using Amicon® Ultra-15 centrifugal filter unit  with a 30 K molecular weight cut-off. Approximately 1 mL of the concentrated enzyme preparations were purified by gel filtration using a GE 16/600 Superdex 200 pg. column . Purified globupain and substitution variants were stored in 20 mM HEPES, 150 mM NaCl, 0.1% CHAPS, pH 7.5 at 4°C.2.6.2, respectively (activation buffer). To investigate if globupain could in-trans activate, 10 μg of activated WT globupain was mixed with 10 μg of inactive C185A variant. The number and size of cleavage products were assessed by visualization of protein bands on 8%–16% SurePAGE precast gels (GenScript) using MES SDS running buffer (GenScript) in a Bio-Rad Mini-PROTEAN Tetra Cell . For sample preparation, 4′ lithium dodecyl sulfate (LDS) sample buffer (GenScript) with 2-mercaptoethanol was mixed with the protein sample, followed by denaturing at 95°C for 10 min. Gels were stained with InstantBlue™ ultrafast protein stain , and the size of bands was indicated by broad multi-color pre-stained protein standard (GenScript). Edman sequencing was performed on a Shimadzu PPSQ-53A at the Iowa State University Protein Facility, United States.For activation of globupain and substitution variants , purifie2.7.4HCO3 in 50% acetonitrile for 10 min each time. The gels were then dried completely in a Savant Speed Vac Plus AR . A mixture of 10 mM TCEP and 25 mM iodoacetamide in 25 mM NH4HCO3 was added to cover the gel pieces and this reaction proceeded in the dark for 1 h. Gels were then washed with 25 mM NH4HCO3 and dehydrated with 25 mM NH4HCO3 in 50% acetonitrile. Samples were then dried in a Savant Speed Vac Plus AR (Thermo Fisher Scientific) before addition of 12.5 ng/uL trypsin in 25 mM NH4HCO3 for peptide digestion. Following a 10 min incubation at 4°C, the samples were covered in 25 mM NH4HCO3 and the digestion proceeded at 37°C for 20 h. The supernatant was then transferred to a clean tube and the remaining peptides were extracted from the gel by addition of 50% acetonitrile, 5% formic acid. The extracted digests were then dried and resuspended in 0.1% formic acid to prepare for C18  ZipTip desalting.C18 columns were washed with methanol and spun for 45 s at 3,500 × g. Columns were then cleaned and equilibrated with 50% acetonitrile, 0.1% formic acid and 0.1% formic acid in water, respectively. Samples were then loaded onto columns and spun for 2 min at 2,000 × g. Samples were washed with 0.1% formic acid and spun at 3,500 × g for 45 s. Peptides were eluted from C18 with 50% acetonitrile, 0.1% formic acid by spinning at 3,500 × g for 45 s. Samples were dried in a Savant Speed Vac Plus AR (Thermo Fisher Scientific) and stored at −80°C until they were prepared for mass spectrometry.Following staining with InstantBlue™ ultrafast protein stain , the gel bands  were exc2.8.E. coli (GCA_000022665.2) and globupain proteome  analysis was performed using a Superdex 75 10/300 GL prepacked column connected to ÄKTA pure 25 chromatography system . The column was equilibrated with a 50 mM potassium phosphate buffer (pH 7.0), 150 mM NaCl and then loaded with a 500 μL sample of globupain protein (1 mg/mL). The flow rate of the run was adjusted to 0.5 mL/min, and the absorbance was measured at 280 nm . For the experiment, the column was calibrated with proteins of known molecular weight: alcohol dehydrogenase (tetramer), 146,800; bovine serum albumin, 66,000; ovalbumin, 43,000; trypsin inhibitor, 22,000; and cytochrome C, 12,400 . Dextran blue 2000 (Cytiva) was used to determine the column void volume.2.10.3), and viscosity , were measured at 20°C using Anton Paar DMA 5000 density meter and Lovis 2000 ME viscometer. Protein partial specific volume (V-bars) was estimated at 0.7309 mL/g using SEDNTERP software . The results were plotted using GUSSI graphical program , equipped with AN 60Ti 4-hole rotor and 12 mm path length, double-sector charcoal-Epon cells, loaded with 400 μL of samples and 410 μL of buffer . The experiments were conducted at 20°C and 50,000 rpm, using continuous scan mode and radial spacing of 0.003 cm. Scans were collected in absorbance, in 4 min intervals at 280 nm. Data were analyzed using the “Continuous c(s) distribution” model of the SEDFIT program , with a  program .2.11.m) of proteins was determined by thermal unfolding with a temperature gradient between 20°C and 110°C at a ramp rate of 1°C/min. Thermal unfolding was measured by tryptophan and tyrosine fluorescence change at 330 and 350 nm emission wavelengths. All measurements were performed in triplicates.Thermostability of the inactive globupain and its activated form were assayed by nanoscale differential scanning fluorimetry (nanoDSF). Measurements were performed with Prometheus NT.48 instrument  and PR.ThermControl software using standard grade capillaries. Before measurement, the capillaries were sealed with a sealing paste according to the manufacturer recommendations. The results were further analyzed with PR.StabilityAnalysis software. Thermostability of globupain zymogen at 0.4 mg/mL concentration and its activated form were assayed in 20 mM tri-sodium citrate (pH 5.5) buffer with 150 mM NaCl and 20 mM HEPES buffer (pH 7.5), 500 mM NaCl with 25 mM imidazole, respectively. Melting temperature , respectively. The gelzan™ CM plate assay was prepared by autoclaving 1.5% gelzan™ CM (Sigma-Aldrich) dissolved in 20 mM tri-sodium citrate (pH 5.5) buffer with 150 mM NaCl. The casein powder (Sigma-Aldrich) was dissolved in 20 mM tri-sodium citrate dihydrate (pH 5.5), 150 mM NaCl. NaOH was added until the casein was fully dissolved in the solution and then autoclaved at 115°C for 10 min. Casein was added to the gelzan™ CM solution at a final concentration of 1.0%. The casein gelzan™ CM solution was poured into sterile glass Petri dishes and set to harden. Wells were made by punching holes in the plates using an inverted sterile 1 mL pipet tip. To test for proteolytic activity, 60 μL of activated globupain at 0.7–1.0 mg/mL was added to wells and incubated overnight at 75°C. Clearance zones would indicate caseinolytic activity.2. The caseinolytic activity was measured by running a time-resolved fluorescence read at 60°C, measuring fluorescence intensity every 20 s for 100 cycles. Fluorescence was measured with excitation wavelength 485 nm and emission wavelength 530 nm using an EnSpire™ 2300 Multilabel Reader . All measurements were done in triplicates and baseline corrected using GraphPad Prism 9.1.0.When the proteolytic activity was assessed using the EnzChek™ Protease Assay Kit (Thermo Fisher Scientific), 20 mM tri-sodium citrate (pH 5.5) buffer with 150 mM NaCl was used to dilute the 1.0 mg/mL stock solution of BODIPY FL casein to 10 μg/mL. An aliquot of the activated enzyme (0.15 μg) was then added to the reaction mixture  comprising 12.5 μL of 10 μg/mL BODIPY FL casein working solution and 77.5 μL of 20 mM tri-sodium citrate (pH 5.5) buffer with 150 mM NaCl, 10 mM DTT, and 1 mM CaCl2.13.2, pH 5.5, and mixed 1:1 with globupain such that the final concentration in the assay was of 2.9 μg/mL enzyme and 50 μM substrate. Assays were performed in triplicate wells of a black 384-well plate (Thermo Fisher Scientific). Fluorescence was measured at 50°C over 1 h at excitation 360 nm and emission 460 nm on a BioTek Synergy HTX Multimode Reader . The reaction rate was calculated as the maximum velocity over 12 sequential readings and means with standard errors were calculated. A Welch’s ANOVA and Brown Forsythe ANOVA were performed to calculate significances in GraphPad Prism 9.1.0. With Boc-QAR-AMC substrate, the Michaelis Menten kinetics was assessed at final concentrations ranging from 0 to 400 μM, and a Michaelis–Menten curve was fitted in GraphPad Prism 9.1.0  reporter group were screened for hydrolysis by globupain. These substrates consisted of Ac-VLTK-AMC, Ac-VLGK-AMC, Ac-VLVK-AMC, Ac-IK-AMC, Ac-YK-AMC, Ac-LK-AMC, Ac-LETK-AMC, Ac-IETK-AMC, Ac-AEIK-AMC, Ac-AIK-AMC, Boc-LRR-AMC (R&D Systems S-300), N-Benzoyl-FVR-AMC (Bachem I-1080), z-RR-AMC (Sigma C5429), Ac-RLR-AMC (AdipoGen AG-CP3-0013), Boc-QAR-AMC , z-VVR-AMC , Pyr-RTKR-AMC  where the N-terminal blocking groups Ac, Boc, z, and Pyr, correspond to acetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, and pyroglutamyl, respectively. Substrates containing K-AMC were synthesized by Dennis Wolan, The Scripps Research Institute, La Jolla, California and purified to >95%. All substrates were stored at −20°C as 10 mM stocks in DMSO. Substrates were diluted to 100 μM in 20 mM tri-sodium citrate dihydrate, 150 mM NaCl, 2.5 mM DTT, and 1 mM CaClsm 9.1.0 .2.14.4 and 0.1 M citric acid following the McIlvaine’s buffer system  classified as an uncharacterized genome within the genus Archaeoglobus . The putative polypeptide comprised 481 amino acids with a 21 amino acid N-terminal signal peptide (C. histolyticum), distapain of 27.3% (Parabacteroides distasonis), PmC11 of 24.2% (Parabacteroides merdae) and thetapain of 26.9% (Bacteroides thetaiotaomicron). Sequence alignments (177), including catalytic H133 and C179, were conserved between both structures  cells, with almost 100% of the total recombinant protein as soluble enzyme (132/C185). H132 of the light chain is responsible for deprotonating the neighboring C185 in the heavy chain, which then promotes its nucleophilic attack on the substrate. For globupain, we found that substitution variant C185A generated by site-directed mutagenesis cannot be activated  occurred between K144 and G145 within the sequence LPPIK*GVCWD  and triple (K137A/K139A/K144A) mutants failed to activate into the processed form . This corresponds to a 332 amino acid protein fragment with estimated Mw of 37.67 kDa using the ProtParam tool  with an N-terminal acetyl group (Ac) and a C-terminal AMC reporter group. These substrates were chosen as the P1 residue corresponds to the N-terminal auto-activation site of globupain. For PmC11, Ac-VLTK-AMC was most efficiently cleaved, followed by Ac-VLGK-AMC. However, the substitution of the P2 residue for a hydrophobic Val side chain ablated substrate turnover by PmC11. All three substrates were tested against globupain and found them to be cleaved at a similar rate  among the new 7 substrates occurred between Ac-AIK-AMC (the most efficient) and Ac-YK-AMC (least efficient). Activity with Ac-AIK-AMC was also statistically significantly increased (p < 0.01) compared to Ac-AEIK-AMC. We hypothesized that the broad enzymatic activity of globupain for degrading casein may be partially due to cleavage following a structurally similar amino acid, arginine. Therefore, fluorogenic substrates were examined with Arg as the P1 residue  = 42.85, p < 0.05, R2 = 83.18%. A follow-up post hoc Tukey’s Honest Significance Difference Test (HSD) indicated that pH 7.1 had a stronger effect on decreasing RFU over time relative to the effect measured on RFU at pH 5.5, p < 0.05  are exposed to proteolytic cleavage by neighboring proteases. This kind of activation is known to occur for several C11 enzymes such as thetapain (in-cis (Archaeoglobus (Bacillus species and in the same range as of proteases from (hyper)thermophilic archaea , OQ718499 (https://www.ncbi.nlm.nih.gov/nuccore/OQ718499.1/), SAMN04111445 (https://www.ncbi.nlm.nih.gov/biosample/SAMN04111445/), and JARQZL000000000 (https://www.ncbi.nlm.nih.gov/nuccore/JARQZL000000000.1/).The datasets presented in this study are deposited in the NCBI online repository, under accession numbers PRJNA296938  through the Center for Excellence in Geobiology (grant #179560), the KG Jebsen Foundation, the Trond Mohn Foundation, the University of Bergen through the Centre for Deep Sea Research (grant # TMS2020TMT13), the CAPES Foundation (grant # 88887.595578/2020-00 and 88887.684031/2022-00), UFMG intramural funds, the RCN-funded DeepSeaQuence project (project #number 315427), and Norway Financial Mechanism through the National Science Center (Poland) GRIEG1 grant: UMO-2019/34/H/NZ2/00584. BH was funded by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-nitrosodiethylamine (NDEA) is a potential carcinogen known to cause liver tumors and chronic inflammation, diabetes, cognitive problems, and signs like Alzheimer’s disease (AD) in animals. This compound is classified as probably carcinogenic to humans. Usual sources of exposure include food, beer, tobacco, personal care products, water, and medications. AD is characterized by cognitive decline, amyloid-β (Aβ) deposit, tau hyperphosphorylation, and cell loss. This is accompanied by neuroinflammation, which involves release of microglial cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin 1β (IL-1β), by nuclear factor kappa B (NF-κB) upregulation; each are linked to AD progression. Weak PI3K/Akt insulin-signaling inhibits IRS-1 phosphorylation, activates GSK3β and promotes tau hyperphosphorylation. Metformin, an antihyperglycemic agent, has potent anti-inflammatory efficacy. It reduces proinflammatory cytokines such as IL-6, IL-1β, and TNF-α via NF-κB inhibition. Metformin also reduces reactive oxidative species (ROS) and modulates cognitive disorders reported due to brain insulin resistance links. Our study examined how NDEA affects spatial memory in Wistar rats. We found that all NDEA doses tested impaired memory. The 80 µg/kg dose of NDEA increased levels of Aβ1-42, TNF-α, and IL-6 in the hippocampus, which correlated with memory loss. Nonetheless, treatment with 100 mg/kg of metformin attenuated the levels of pro-inflammatory cytokines and Aβ1-42, and enhanced memory. It suggests that metformin may protect against NDEA-triggered memory issues and brain inflammation. N-nitrosodiethylamine (NDEA) is an organic compound and a member of the N-nitrosamines, which refers to any molecule containing a nitroso functional group . NitrosaAD is the most common neurodegenerative disease and is clinically manifested by a progressive deterioration in cognition and memory as well as progressive behavior changes . One of Metformin belongs to the biguanide class of drugs. It is the first-line treatment for T2DM and is the most prescribed oral anti-diabetic medication worldwide. The key mechanism through which metformin reduces blood glucose levels is by inhibiting liver glucose production and augmenting glucose utilization ,34. The In this study, we investigated the impact of exposure to different doses of NDEA on spatial memory, hippocampal Aβ levels, and proinflammatory cytokines such as TNF-α and IL-6 in rats. We examined the effect of metformin on memory impairments as well as Aβ1-42 and proinflammatory cytokines induced by NDEA.w/v) was used for dilutions and was stored in the dark. Metformin hydrochloride 200 mg (1115-70-4) was dissolved in NaCl0.9% (w/v) before use.NDEA and metformin were obtained from Sigma Chemical Co. . NDEA (442 687) ampulla 1 mL (1000 mg/mL); NaCl0.9% . Four groups received different injections of NDEA at doses of 20 µg/kg (group 1), 40 µg/kg (group 2), 60 µg/kg (group 3), and 80 µg/kg (group 4). These were intraperitoneal (i.p.) injections that were administered every 48 h on days 1, 3, and 5 (3 n = 8 in each group) were randomly assigned to three groups as follows: (1) NDEA 80 μg/kg i.p.  . It consTraining trials: The animals underwent four daily training trials for four consecutive days. Each trial involved placing the animals in the pool facing the pool wall in one of the selected quadrants (the starting position varied daily). The rats had a maximum of 60 s to locate the hidden platform and 20 s on the platform. The interval between the trials was 20 s. Rats unable to locate the platform within 60 s were manually guided to it and held there for 15 s. The time taken to reach the platform (escape latency) was measured as an indicator of spatial learning.Probe trial: On the fifth day, the platform was removed. The animals were placed in the pool from the quadrant opposite the training quadrant and allowed to swim freely for 60 s. The time spent in the target (SW) quadrant was recorded as a measure of spatial memory. The trials were recorded and analyzed using SMART v3.0.02 Harvard apparatus software.On the 14th day, rats from the second experiment group were euthanized with pentobarbital (50 mg/kg i.p.) and decapitated. Their brains were quickly removed and the hippocampus was dissected. The tissue was frozen at −80 °C until further analysis. The tissue was then homogenized in 10 wet-weight volumes of tris buffer saline (pH 8.0) containing protease and phosphatase inhibitors. The resulting lysates were centrifuged and the supernatants were stored at −80 °C for the ELISA assay.® ELISA (DAB142). The measured values were expressed as amounts per total protein. The assays were conducted as described in the instructions for use. Briefly, standards, controls, and hippocampal samples were added to the wells and incubated at 37 °C for 1.5 h. Biotin-detection antibody was added and incubated for 60 min at 37 °C. The plate was washed thrice using a wash buffer in each well and soaked for 1–2 min. The solution was added and incubated at 37 °C for 30 min. The plate was then washed five times as previously. TMB substrate was added, and the plate was incubated in the dark at 37 °C for 15–30 min. Finally, 50 μL of stop solution was added. The optical density of each well was read at 450 nm within 20 min.TNF-α and IL-6 were measured using a Rat ELISA kit from Invitrogen Co.  . The soluble Aβ1-42 levels were determined using the R&D Systems Quantikinep-value of <0.05 was significant. A normal distribution was confirmed with the Shapiro–Wilk test. We conducted a two-way repeated-measure ANOVA followed by Bonferroni’s post hoc test to compare the acquisition data from the MWM test. A one-way ANOVA followed by Tukey’s post hoc test was used to analyze the percentage of time spent in the target quadrant in the MWM test as well as Aβ1-42, TNF-α, and IL-6 levels. The correlation between memory performance, Aβ1-42, and proinflammatory cytokines was assessed using Pearson’s correlation coefficient.All results were analyzed using GraphPad Prism 8.0 software . Data are expressed as the mean ± standard error of the mean (±SEM). A p < 0.0001] for escape latency and  for traveled distance. The ability of animals to locate the hidden platform progressively decreased during the four days of the acquisition phase in the MWM, except the 20 µg/kg group. The differences in escape latency and traveled distance during training days 2–4 for all groups under treatments by NDEA were statistically significant compared with the control group, with  on days 3 and 4, as it was in the NDEA 80 µg/kg group on days 2, 3, and 4  = 8.535, 3, and 4 . Compareays 2, 3 . The swiays 2, 3 C, suggesp= 0.0009] less time in the target quadrant, compared to the control group (43.8 ± 2.6 m). Times of the treatment groups were: NDEA 20 µg/kg, 35.9 ± 3 m; NDEA 40 µg/kg, 24.1 ± 4.3 m; NDEA 60 µg/kg, 20.2 ± 4.6 m; and NDEA 80 µg/kg, 18.2 ± 5.1 m. These results indicated significant impairment of learning and formation of memory reference with NDEA treatment  = 7.354, reatment D.p = 0.0009], as it was in traveled distance . The NDEA 80 µg/kg group demonstrated a significantly longer escape latency and traveled distance on days 2 and 3, compared to the control group (p > 0.05] (The effect of metformin (100 mg/kg) on cognitive deficits induced by NDEA (80 µg/kg) was tested using the MWM. The spatial learning skills of animals were assessed with four days of hidden platform tasks. A repeated-measure two-way ANOVA followed by a post hoc Tukey test demonstrated a change in the escape latency and the traveled distance during training days 2, 3, and 4 for the NDEA 80 µg/kg + metformin 100 mg/kg group. Between this group and the NDEA 80 µg/kg group, the difference in escape latency was statistically significant  C.p = 0.0035) than the control group. The NDEA 80 µg/kg + metformin 100 mg/kg group remained longer in the target quadrant  than the NDEA 80 µg/kg group  = 1874; p < 0.0001). Treatment with metformin led to a partial recovery (p < 0.05) in the NDEA 80 µg/kg group compared to the control.On the fifth day, the final day of the experiment, the hidden platform was removed to evaluate the memory of the trained rats. p < 0.0001; 103%) than in the control group. Treatment with metformin significantly restored Aβ1-42 levels  in the hippocampus in comparison to the NDEA 80 µg/kg group (p < 0.001) and the control group . Metformin significantly prevented a NDEA-induced rise in TNF-α in the hippocampus   compared to the control group. Metformin significantly decreased IL-6 levels in the hippocampus of NDEA-treated rats   B. The le01; 21%) C.p < 0.01), TNF-α , and IL-6  levels and the consolidation of memory in rats treated with NDEA 80 µg/kg group (p < 0.0001). IL-6 did not show a relationship with Aβ1-42   A,B.In this study, we investigated whether NDEA treatment , sub-mutagenic doses of NDEA have been linked to the development of insulin resistance in the brain. Tong et al. demonstrated that NDEA 20 μg/kg i.p. , and transforming growth factor-beta (TGF-β). This is accompanied by the activation of microglia and astrocytes around Aβ plaques ,56,57. AFurthermore, our results were consistent with the neuroprotector and anti-inflammatory effects of metformin observed in numerous other studies. In a mouse model of Parkinson’s disease, metformin (150 mg/kg) reduced the amount of microglia, proinflammatory cytokines , and inducible nitric oxide synthase (iNOS) . In AD AA significant limitation of our study was the inability to provide a more specific molecular data presentation of the signaling pathways mentioned above. Despite our best efforts, this limitation resulted in an unavoidable absence of molecular results. This omission limited our ability to obtain detailed conclusions about molecular interactions within our study data. To mitigate this, we attempted to describe broader findings that emerged from the molecular data in a simplified and accessible manner. Our research findings suggested that learning and memory impairment induced by NDEA was associated with high levels of Aβ1-42, TNF-α, and IL-6 in the hippocampus, and these effects were recovered by metformin. Hence, metformin could protect against NDEA-induced brain damage, indicating its potential as a neuroprotective and anti-inflammatory treatment in AD. Based on the existing literature, our results can be explained as a demonstration that metformin could improve memory dysfunction by mitigating proinflammatory cytokines production through the inactivation of NF-κB. This action could potentially enhance the insulin signal sensitivity via the PI3K/AKT/mTOR/Bace-1 pathway and suppress the production of Aβ1-42. Future research will focus on the NF-kB signaling pathways regulating TNF-α and Aβ.