Bacillus subtilis” (B. subtilis) microorganisms can effectively repair structural and non-structural cracks caused at the nano- and microscale. By creating an effective immobilization strategy in a coherent manner, the primary challenge regarding the viability of such microbes in a concrete mixture atmosphere has been successfully fulfilled. The iron oxide nanoparticles were synthesized. The examined immobilizing medium was the iron oxide nanoparticles, confirmed using different techniques . By measuring the average compressive strength of the samples (ASTM C109) and evaluating healing, the impact of triggered B. subtilis bacteria immobilized on iron oxide nanoparticles was examined. The compressive strength recovery of cracked samples following a therapeutic interval of 28 days served as a mechanical indicator of the healing process. In order to accurately correlate the recovery performance as a measure of crack healing duration, the pre-cracking load was set at 80% of the ultimate compressive stress, or “f c,” and the period of crack healing was maintained at 28 days. According to the findings, B. subtilis bacteria greatly enhanced the compressive strength and speed up the healing process in cracked cement concrete mixture. The iron oxide nanoparticles were proven to be the best immobilizer for keeping B. subtilis germs alive until the formation of fractures. The bacterial activity-driven calcite deposition in the generated nano-/micro-cracks was supported by micrographic and chemical investigations .For the creation of healable cement concrete matrix, microbial self-healing solutions are significantly more creative and potentially successful. The current study investigates whether gram-positive “ Different types of precursors are used by scientists to obtain CaCO3. The most common precursors include amino acid salt [2 to CaCO3, which is basically produced when water comes into contact with the urea or salt of the amino acid given in Equation (1). When there is Ca+2 in the surrounding atmosphere, the negatively charged bacterial cell membrane will pull positive charges from the surroundings after the Ca+2 is in the process of demineralization. Calcium ions (Ca+2) will ultimately be collected on the surface of the cell, as shown in +2 ions integrate with the CO3−2 ions, resulting in CaCO3 precipitation, as shown in Equation (2).Concrete has become one of the world’s most widely utilized substances due to its unique properties, which include good compressive strength, proximity, adaptability, economically, suitability with reinforced steel bar, flame retardation, superior caloric weight, and its ability to be cast in specific forms and dimensions ,2. Concrcid salt ,17 and ucid salt ,19. The 2 to CaCO3 by non-methylotrophic methanogenesis, aerobic photosynthesis, and anaerobic photosynthesis using different bacteria in a variety of environments. MICP has been discovered in the formation of calcium carbonate in a variety of geological environments, including soils, limestone caves, oceans, and soda lakes [Calcium carbonate is made outside of the cell membranes of bacteria via autotrophic and heterotrophic metabolic processes. The autotrophic microbes convert the COda lakes . Biotic da lakes . Using Mda lakes ,19,22,23da lakes ,17,19. 3) were also discussed in the paper.The number and quality of induced minerals have a significant impact on the efficacy of MICP processes ,24. For 3.6H2O, FeCl2.4H2O, sodium hydroxide, MnSO4.H2O (Sigma Aldrich > 99% pureness), ethanol (Merck > 99–100% purity). Reagents were used during the synthesis, characterization, and reaction study, with no further purification.The bacterial concrete used in this research consisted of the commercial calcium lactate, purchased from Sigma-Aldrich, Schnelldorf, Germany. The chemicals included FeClBacillus subtilis was isolated from soil using a streak plate technique. Ordinary Portland cement (Grade 53) was purchased from Fauji Cement Company  . The iron oxide nanoparticles were synthesized in the laboratory using a chemical precipitation method. Margalla coarse aggregates were used in this research. The calcium lactate used was a high purity product purchased from Sigma-Aldrich, Germany. The urea, calcium chloride anhydrous, and ammonium hydroxide were purchased from Daejung, Siheung si, Korea. The yeast extract, nutrient broth, and nutrient agar were purchased from Oxoid . Deionized distilled water was produced in the laboratory using a B114 deionizer.For the casting of the bacterial concrete, the bacteria 3.6H2O in distilled water was designated as solution A. In addition, a second solution B was generated by the addition of 2.1 g of FeSO4.7H2O in distilled water. Under intense stirring, solution B was added dropwise to solution A and heated up to 80 °C. Soon after, the appearance of the color black was a sign of the IONPs. The nanoparticles were filtered and washed with ethanol and distilled water thrice. The precipitates were then dried in an oven  overnight at 80 °C. The chemical precipitation method was used to synthesize the IONPs. A schematic diagram is shown in −1 until reaching 800 °C from the ambient temperature. The results were reported as the percent weight loss against the relative temperature. Functional groups in the synthesized nanomaterial were identified and compared using an Alpha Bruker, FTIR Spectrophotometer . The nanomaterials were pressed into pellets and scanned at 4000 to 400 cm−1. The procedure for obtaining the FTIR spectra was as follows. The synthesized sample of the IONPs was heated for 24 h at 80 °C in an oven in a controlled environment. The obtained IONPs were placed in a sample container. Spectrum analysis software was used to acquire the infrared spectrum. For analysis of the white healing precipitate, samples were extracted from the broken pieces of bio-concrete. The white precipitated samples of 30 mg were dried first in an oven at 50 °C and then ground into a fine powder < 5 μm in size. The characterization of the IONPs was crucial to better understand the physio-chemical properties. The performance of these nanoparticles can be best explained based on these properties as a nanopellet for the encapsulation of bacteria when put into the concrete mix during casting. The information derived from different characterization techniques can be helpful for understanding the relationship between the physicochemical characteristics and their efficiency as carriers of bacteria. The thermal stability of IONPs was also checked. As IONPs were added to the concrete mix, the temperature within the concrete raised to 80–90 °C due to hydration reactions . TherefoThe crystalline sizes of the IONPs were measured using X-ray diffraction . The pattern of XRD was scanned at a scan rate of 2° per min from 20–80° using a Cu Kα radiation source. The IONP samples were collected in fine powder form. For the characterization of the healing material, the test samples were collected from the outer surface of the bio-concrete. Moreover, the healing material from remainant pieces of healed bio-concrete samples after the compression test were obtained and then ground into a fine powder < 5 μm. The crystalline size was estimated using the Scherrer equation .(3)D=KλSurface morphology was conducted with scanning electron spectroscopy using a JEOL, JSM-6510LA, Tokyo, Japan. Scanning electron microscopy  was usedBacillus subtilis was selected for this study. Bacillus subtilis is obtained, isolated and grown from the soil at COMSATS University in Abbottabad, Pakistan. The soil sample was picked from a fresh vegetable dump area. A weight of 5 g of the soil sample was taken in 25 mL TBS, suspended, and diluted to the specific dilution factor. It was then plated on TSA and incubated for 24 h at 37 °C. The method that is represented in Bacillus subtilis from the soil by obtaining the grown colony and using it for identification. The microorganism 4)2SO4 or KNO3, 0.8 g K2HPO4, 0.2 g KH2PO4, 0.5 g MgSO4 and 7H2O, 0.05 g CaSO4.7H2O, 0.01 g FeSO4.7H2O, and 1.2 g agar. Another was added to water to 1 L, and the pH scale was adjusted to seven. The square plate’s (petri dish) was incubated at 37 °C. White, round, sleek, and glossy colonies 1–3 mm in diameter developed on the nitrate (KNO3) medium in 24 h. However, not all strains use nitrate, so the advice is to use ammonium ion ((NH4)2SO4) medium in parallel. Rod-shaped colonies were detected by their look and the general giant cells of this species were confirmed microscopically. The details of the isolation of the bacteria from soil are shown in The technique used for the isolation of bacteria was the streak plate technique. It started with plating heat-treated soil suspensions on aldohexose mineral base agar in a cone-like flask with 10 g glucose, 1 g (NHIdentification was performed using a polymerase chain reaction (PCR) test after extracting the deoxyribonucleic acid (DNA) and matching it. A morphological investigation was also conducted using a microscope for identification. After identification, the bacteria grew on a plate using the streak plate technique. For the streaking technique, first, the distilled water in the required volume, and nutrient agar and petri plates were placed for autoclaving (121 °C for 15 min), and then add nutrient agar (28 g/L) to the distilled water, and finally apply on the petri dish. After applying agar media on the plates in a laminar flow bath (UV light-treated for 10 min) the streaking petri plates were then placed in a static incubator (at 37 °C for 24 h) to verify the contamination. Afterward, the colonies of the bacteria were removed using a wire loop, first made red hot by using a spirit lamp in laminar flow, and streaked on the agar media plate. After the streaking, the plates were placed in a static incubator at (37 °C for 24 h) and checked for the growth of bacteria without contamination for further use in the process. The growth of the bacteria is shown in w/v), glucose (0.5% w/v), and yeast extract (0.05% w/v) were incubated at 37 °C (120 rpm and 24 h) to rehydrate the bacterial strains in the nutrient broth media. After 24 h of growth of the bacteria in a shaking incubator, the colonies were counted using a UV-Vis spectrophotometer (OD600 nm). First, a blank sample of agar media was obtained (3 mL) in a glass cuvette and placed in the spectrophotometer (OD600 nm) set at zero for reading. Then the cuvette was filled with 3 mL of grown agar culture and placed in the spectrophotometer. A reading was taken and then put into Ramachandran’s equation for counting the bacterial colonies.After obtaining the true strain grew, a colony was chosen to be spread in a cone containing nutrient broth. The media containing peptone  was placed in a water bath (80 °C for 10 min). The spore suspension was obtained by performing centrifugation for 15 min at a speed of 3000 rpm. The obtained spores were then encapsulated in immobilization media to encase IONPs in concrete during casting. IONPs (250 µg/mL) were obtained and dispersed in distilled water using sonication for 15 min at 35 °C in a sonicator to create pores for the bacteria encapsulation. Then the mixture was transformed into a suspension containing bacterial cells for immobilization. For the bacteria to be encased in concrete during casting, food consisting of the nutrients calcium chloride anhydrous (40 g/L), urea (65 g/L), and yeast extract (2 g/L) was dissolved in distilled water. Then, the solution was inoculated with the immobilized bacteria IONPs, and the bacterial food was mixed and stirred for 2 min and gradually added to the concrete mixture.3, 106, and 109 cells of true bacteria Bacillus subtilis per milliliter of water. Four concrete mixes—a control concrete and three concrete mixes, including the different concentrations of bacteria solution  cells/mL—were cast in molds of 150 mm × 150 mm × 150 mm.The details of the bio-concrete cast in this study are listed in Bacillus subtilis is considered to be a harmless organism as it does not possess traits that can cause a disease or that are harmful to human beings [The bacterium n beings . Moreoven beings . The inan beings , this ban beings .The normal consistency of cement was obtained to determine the amount of water to be added to the cement for a paste of normal solidity. Ordinary Portland cement (OPC) was used as a binder in all concrete types, and X-ray fluorescence (XRF) was used to obtain the chemical composition of OPC. The physical and chemical properties of the binder are mentioned in 3, 106, and 109 cells of Bacillus subtilis per milliliter of water were used. Concrete cube specimens 150 mm × 150 mm × 150 mm in size were hardened to develop a 28-day compressive strength of 20 MPa. At 28 days, the specimens were tested for compressive strength using a compression testing machine. Avoiding any shocks, loads were applied at an increasing rate of approximately 140 kg/cm2/min until the specimen could not sustain the load. The maximum load was recorded, and the compressive strength was calculated by dividing the maximum load by the size of the cross-sectional area. This test was conducted on four specimens and the average value was taken. The test was conducted as per ASTMC 642-97 to work out the inflated resistance to water penetration in the concrete. The control and bacterial concretes were cast in cubic molds 150 mm in size. The square specimens were cured for 28 days. To determine the water absorption percentage for both the control and bacterial concrete, the specimens were placed in a water tank and cured for 28 days. For the bacterial concrete samples with varying concentrations of encapsulated IONP bacteria, both the pre-cracking and post-cracking wet weights were recorded using a scale, as shown in 3, 106, and 109 cells/mL of potable water.A slump test was performed to determine the consistency and workability of the cement concrete. A concrete is determined to be viable if it can be simply mixed, placed, compacted, and finished. A viable concrete should not show any segregation or damage. The control concrete and microorganism concrete mixtures were designed to have a slump of 25–75 mm. For the microorganism concrete mixture, cell concentrations of 10The ultra-sonic pulse velocity test as shown in 3, 106, and 109 cells per milliliter of blending water with Bacillus subtilis. All the ingredients needed for the M20 concrete mixture were weighed and mixed. Further, the specimens were cast instantly with the admixture. Cracks were introduced until they were visible and measurable within the cube specimen by applying loads using a compression testing machine. The concrete was removed before the final setting of the concrete and a specified crack was clearly seen within the specimen. Then the specimen was placed for healing in a natural process tank and images were taken to ascertain the healing of the crack for a healing evaluation period of 28 days of immersed curing. The presence of white precipitates during healing evaluation period indicated that the crack was healing well.The microorganism concrete cube specimen was cast with microorganism cell concentrations of 10The mix proportion ratios for the M20 ordinary concrete cubes were finalized as shown in 3O4 compositional components generated through co-precipitation synthesis, the Fe had a value of 53.80%, while the O had a value of 32.30%. These data show that the synthesis products and samples were pure and had excellent stoichiometry. The SEM-EDX showing an FeO percentage of 89.55%  reflect that the sample was pure, and no other impurity peaks were observed, as shown in The SEM analysis shows that the IONPs particles were small porous particles, rounded and spherical, as shown in 3O4 nanoparticles that the characteristic absorptions of the Fe-O bond are at 530 cm−1 and 634 cm−1. Both the peaks at 530 cm−1 and 628 cm−1 in −1 confirms the existence of carbon. The results in the literature [3O4 nanoparticles.It can be seen in the FTIR spectra of the prepared Feterature  also con3O4 samples displayed six typical peaks at values of 30, 35.4, 43, 57, 62.67, and 66.37, which correspond to the (238), (351), (195), (230), (243), and (197) crystal planes, as can be seen in 3O4 or Fe. Furthermore, the peaks are quite wide, indicating the creation of very tiny particles in the nano-scale region [3O4 nanoparticles had a spinal structure with no distinctive impurity peak. Similar findings were discovered in the literature [The crystalline phases of the products were determined using XRD measurements. The Fee region . The aveterature ,35.3O4. The first weight loss of less than 1% up to 200 °C can be attributable to the existence of residual physisorbed water-based solution solvents. A weight loss of roughly 2% was observed for nanoparticles generated by chemical precipitation at temperatures ranging from 300 °C to around 800 °C. This indicates that unreacted sodium acetate existed within the nanoparticles.The thermal stability of the IONPs was also determined. As IONPs were added to the concrete mix, the temperature within the concrete increased to 80–90 °C due to hydration reactions . Therefo9 cells/mL (BC-9) shows more gain in the compressive strength compared to the bio-concretes with 106 (BC-6) and 103 (BC-3) cells/mL, respectively, as shown in 9 cells/mL healed wider cracks than concrete with a bacterial concentration of 106 cells/mL. In addition, the results demonstrate that with increases in the bacterial concentration, the calcium carbonate precipitation also increased.The compressive strength was determined at 28 days after casting the control M20 concrete mix. After 28 days of immersed curing, the average result of the compressive strength was recorded at 19.64 MPa. The average compressive strength of the control mix was considered a reference for comparison with the compressive strength results of the bio-concrete. The compressive strength results of the bio-concrete show an increase in the strength gain compared to the control concrete mix, as shown in 6 cells/mL had no impact on the quality of concrete, according to the UPV test results  being the most common polymorph formed in the bio-concrete samples. A similar outcome can be found in the literature [The XRD analysis of microbially induced CaCOBacillus subtilis cells . The white precipitate deposited by Bacillus subtilis produced extremely distinctive acute stretching bands of calcites from 1800 to 1550 cm−1 [The FTIR analysis of microbially produced calcium carbonate in powder appears to be a useful approach for early identification, as shown in 550 cm−1 ,40. It was established that bacterial immobilization using IONPs was a viable technique for addressing cell vulnerability in the construction of self-healing bio-concrete. The following conclusions can be drawn from this investigation.B. subtilis bacteria to the cement concrete mix, which changed with the concentration of bacteria employed in the cementitious material. The bio-concrete with 109 cells/mL (BC-9) shows a 25.9% increase in compressive strength. The B. subtilis microorganisms have a great potential to produce CaCO3 for healing nano-/micro-scale structural/nonstructural cracks in cementitious composites, as evidenced by the repair of micro-cracks to wider cracks of 1.71 mm and a crack length of 127.0 mm completely. The creation of a protective layer surrounding bacterial spores is responsible for the contradictory behavior of IONPs that contributes to successful healing at older ages. The mechanical method was used in this work to quantify the self-healing of cracks. Additional research employing stress–strain data, as well as fracture origin and development pattern, may be performed in order to better understand the self-healing mechanism. The researchers also recommend expanding the investigation in the future to include complex bio-based analyses to determine the concentrations of consumed and conserved microorganisms at different times.The compressive strength of the samples improved with the addition of


We aimed to identify serologic parameters that correlate with the disease activity of IgG4-related disease (IgG4-RD) in patients with normal and elevated serum IgG4 concentrations, respectively.This retrospective cohort study included 148 patients with IgG4-RD. Patients were categorized into normal (≤201 mg/dL) and elevated (>201 mg/dL) serum IgG4 concentration groups. Disease activity was assessed using the IgG4-RD responder index (RI). The correlations between IgG4-RD RI and serologic parameters  were evaluated in each group, using Spearman’s correlation coefficient.Of the 148 patients with IgG4-RD, 38 (25.7%) and 110 (74.3%) patients were categorized into the normal and elevated serum IgG4 concentration groups, respectively. In the normal serum IgG4 concentration group, IgG concentration was the only serologic parameter that showed a significant correlation with IgG4-RD RI . However, in the elevated serum IgG4 concentration group, ESR , C3 , C4 , IgG4 concentration , IgG concentration , and IgG4/IgG ratio  correlated with IgG4-RD RI. The combination of C3 and IgG4 concentration  had the strongest correlation with IgG4-RD RI in this group.Among the serologic parameters tested, IgG concentration was the only parameter that correlated with IgG4-RD RI in patients with normal serum IgG4 concentrations, whereas multiple parameters correlated with IgG4-RD RI in those with elevated serum IgG4 concentrations. The combination of C3 and IgG4 concentration had the strongest correlation coefficient in the latter group. Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated condition characterized by fibroinflammatory lesions that can affect virtually any organ –3. AlthoIgG4-RD responder index (RI) is a validated disease activity assessment tool widely used for IgG4-RD . The scoIn this study, using a cohort of patients with IgG4-RD, we categorized patients into those with normal serum IgG4 concentrations and those with elevated serum IgG4 concentrations and assessed the correlation between serum IgG4 concentration and disease activity in each group. We also tested other easily accessible serologic parameters, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), complement 3 (C3), complement 4 (C4), IgG concentration, and IgG4/IgG ratio, as potential biomarkers for disease activity in each group.Patients who were newly diagnosed with IgG4-RD between 2011 and 2021 at two referral hospitals were retrospectively included for analysis. All included patients fulfilled the 2019 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for IgG4-RD . Data onWe used the IgG4-RD responder index (RI) as the gold standard of disease activity assessment . All patContinuous variables following normal or non-normal distribution are expressed as mean (± standard deviation [SD]) or median (interquartile range [IQR]), respectively, and categorical variables are expressed as numbers with percentages. For comparison between the two groups , we used the independent two-sample t-test or Mann–Whitney U test for continuous variables following normal or non-normal distribution, respectively. For comparison of categorical variables, the χ2 test or Fisher’s exact test was used. Correlations between IgG4-RD RI and serologic parameters  were evaluated using the Spearman’s correlation coefficient. Correlations were assessed in the normal serum IgG4 concentration and elevated serum IgG4 concentration groups. Multivariable linear regression analysis with backward elimination was conducted to select a combination of serologic parameters that could potentially have a stronger correlation with IgG4-RD RI. All variables that significantly correlated with IgG4-RD RI in the correlation analysis were included in the multivariable model, except for the serum IgG4/IgG ratio, which was excluded due to multicollinearity with serum IgG4 concentration and serum IgG concentration. The variables selected in the multivariable analysis were combined by multiplying each variable by its respective β coefficient, and then summing them. A p-value of < 0.05 was considered statistically significant. All analyses were conducted using SPSS software . Figures were generated using GraphPad Prism .A total of 148 patients with IgG4-RD who fulfilled the 2019 ACR/EULAR classification criteria for IgG4-RD were included for analysis. The median value of the total points of the 2019 ACR/EULAR classification criteria for IgG4-RD was 26.0 (23.0–34.0). The baseline characteristics of the patients are shown in Compared with patients with normal serum IgG4 concentrations, those with elevated serum IgG4 concentrations were older , more commonly had salivary gland , pancreas  and multiple organ involvement (p = 0.019), and had lower CRP levels  Table 2.In the normal serum IgG4 concentration group, serum IgG4 concentration did not significantly correlate with IgG4-RD RI  Figure 1In this study, we found that serologic parameters that correlate with the disease activity of IgG4-RD differ between patients with normal and elevated serum IgG4 concentrations. IgG concentration was the only serologic parameter that correlated with IgG4-RD RI in patients with normal serum IgG4 concentrations. On the other hand, several serologic parameters, including ESR, C3, C4, IgG4 concentration, IgG concentration, and IgG4/IgG ratio, correlated with IgG4-RD RI in patients with elevated serum IgG4 concentrations. This finding is meaningful because it provides better insights into how serologic parameters could be interpreted in regard to disease activity in patients with IgG4-RD who have normal and elevated serum IgG4 concentrations, respectively.In our study population, 25.7% of the patients had normal serum IgG4 concentrations. Similarly, in previous studies, normal serum IgG4 concentrations have been reported in 9.7–48.5% of patients with IgG4-RD . Thus, iAnother significant difference between patients with normal and elevated serum IgG4 concentrations was the lower CRP level in patients with elevated serum IgG4 concentrations. A recent study has shown that patients with higher number of superficial organ involvement  have significantly lower CRP levels than those with internal organ-dominant  involvement . In our Our data revealed that serologic parameters that correlate with the disease activity of IgG4-RD differ between patients with normal and elevated serum IgG4 concentrations. In patients with normal serum IgG4 concentrations, IgG concentration , but not IgG4 concentration , significantly correlated with IgG4-RD RI. In contrast, in patients with elevated serum IgG4 concentrations, multiple serologic parameters including IgG4 concentration  correlated with IgG4-RD RI. The precise role of IgG4 on the disease pathogenesis of IgG4-RD remains unclear; an adoptive transfer model has shown that injection of patient IgG4 in neonatal BALB/c mice results in pancreatic and salivary gland injuries, suggesting a pathogenic role of IgG4 ; while aSimilar to the correlations observed in patients with elevated serum IgG4 concentrations in our study, a previous study consisting of 72 patients with IgG4-RD (68 [94.4%] patients with elevated serum IgG4 concentrations) showed that serum IgG4 concentration, serum IgG concentration, C3, and C4 correlate with IgG4-RD RI . We furtWe used a cut-off of 201 mg/dL to categorize patients into normal serum IgG4 concentration and elevated serum IgG4 concentration groups. Diagnostic criteria for IgG4-RD, established a decade ago, used a value of 135 mg/dL for the cut-off of elevated serum IgG4 concentration . One migOur study had some limitations. First, the possibility of prozone phenomenon in the patients with normal serum IgG4 concentrations cannot be excluded . As thisIn conclusion, we demonstrated that serologic parameters correlate differently with disease activity depending on serum IgG4 concentrations. IgG concentration was the only serologic parameter that correlated with IgG4-RD RI in patients with normal serum IgG4 concentrations, while multiple serologic variables including ESR, C3, C4, IgG4 concentration, IgG concentration, and IgG4/IgG ratio correlated with IgG4-RD RI in patients with elevated serum IgG4 concentrations. In the latter group, a combination of C3 and IgG4 concentration as a composite parameter showed the strongest correlation with IgG4-RD RI. These findings could be useful for a more accurate serologic assessment of disease activity in patients with normal and elevated serum IgG4 concentrations, respectively.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) of Gangnam Severance Hospital (IRB No: 3-2022-0247). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.OCK, M-CP and Y-GK contributed to the conception and design of the study. OCK, M-CP and Y-GK participated in acquisition of data, data analyses and data interpretation. OCK, M-CP and Y-GK wrote the manuscript. All authors contributed to the article and approved the submitted version.Y-GK received grants from the National Research Foundation of Korea (NRF-2021M3A9G1026605).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.


Previous studies have demonstrated that promyelocytic leukemia zinc finger protein (PLZF) promotes the expression of gluconeogenic genes and hepatic glucose output, which leads to hyperglycemia. However, the role played by PLZF in regulating lipid metabolism is not known. In this study, we aimed to examine the function of PLZF in regulating hepatic lipid and glucose homeostasis and the underlying mechanisms. The expression of PLZF was determined in different mouse models with regard to non-alcoholic fatty liver disease (NAFLD). In the next step, adenoviruses that express PLZF (Ad-PLZF) or PLZF-specific shRNA (Ad-shPLZF) were utilized to alter PLZF expression in mouse livers and in primary hepatocytes. For the phenotype of the fatty liver, histologic and biochemical analyses of hepatic triglyceride (TG), serum TG and cholesterol levels were carried out. The underlying molecular mechanism for the regulation of lipid metabolism by PLZF was further explored using luciferase reporter gene assay and ChIP analysis. The results demonstrated that PLZF expression was upregulated in livers derived from ob/ob, db/db and diet-induced obesity (DIO) mice. Liver PLZF-overexpressing C57BL/6J mice showed fatty liver phenotype, liver inflammation, impaired glucose tolerance and insulin sensitivity. On the other hand, hepatic PLZF knockdown in db/db and DIO mice alleviated hepatic steatosis. Of note, we found that PLZF activates SREBP-1c gene transcription through binding directly to the promoter fragment of this gene, which would induce a repressor-to-activator conversion depending on its interaction with SIRT1 in the role played by PLZF in the transcription process through deacetylation. Thus, PLZF is identified as an essential regulator of hepatic lipid and glucose metabolism, where the modulation of its liver expression could open up a therapeutic path for treating NAFLD. Hepatic lipogenesis dysfunction accelerates the pathogenesis of metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). In NAFLD, fatty acids are ectopically accumulated in the liver . NAFLD, via inhibition of SREBP-1c expression , also called ZBTB16 (zinc finger and BTB domain containing 16), is considered a transcription factor that takes part in regulating a number of different biological processes, such as spermatogenesis , stem cee output , the rolvia mediating the SREBP-1c expression. Our study demonstrated that PLZF has a crucial role in the regulation of lipogenesis. This process was dependent on its interaction with SIRT1, which allowed for a repressor-to-activator conversion in the role played by PLZF in the transcription process through deacetylation. Thus, PLZF is found to be a vital regulator of hepatic lipid metabolism, where the modulation of its liver expression may open up a therapeutic path for treating NAFLD.Given what was stated above, we set out a goal to examine whether PLZF is involved in the regulation of hepatic lipogenesis −/−) mice featuring conditional delete of SIRT1 exon four were the result of crossing between mice with floxed alleles of SIRT1 and liver Cre recombinase-expressing mice  or high-fat diet  with free access to water. For adenovirus injection, mice were injected via their tail vein with adenovirus expressing 1) green fluorescent protein (Ad-GFP), 2) PLZF (Ad-PLZF), and 3) short-hairpin (sh) RNA against luciferase (Ad-shCON) or shRNA against PLZF (Ad-shPLZF) as controls (0.5–1.0 × 109 pfu/mouse in 150 μl PBS). In the next step, after 5–7 days elapsed from the infection, the liver and plasma of the mice were retrieved for further analysis. The grouping of all the mice was performed in a random manner, and the researchers performing the experiments were blind to the assignment of the groups and the evaluation of the outcome.The experimental procedures concerning animals received approval from the Animal Care and Use Committee of Zhongshan Ophthalmic Center of Sun Yat-sen University; they were carried out in accordance with all relevant ethical regulations. Male ob/ob, db/db, db/m, and C57BL/6J mice (aged six to 8 weeks) were acquired from the Model Animal Research Center of Nanjing University  and were kept in standard cages. The cages were placed in a specific pathogen-free facility, in which a 12-h light/dark cycle was maintained. The animals were subjected to free access to food and water. Liver-specific SIRT1-knockout  and 5′-CCGGATATCCAGATCCTCTTCAGAGATGAGTTTCTGCTCCACATAACACAGGTAGAGGTACGT-3’ (Reverse) for PLZF or 5′-CCGGGTACCATGGCGGACGAGGTGGCGCT-3’ (Forward) and 5′-CCGCTCGAGGCTTATCGTCGTCATCCTTGTAATCTGATTTGTCTGATGGATAGT-3’ (Reverse) for SIRT1. PLZF- or SIRT1-expressing recombinant adenoviruses were generated following what was described in ref. . PCR wasThe design and synthesis of shRNAs targeting PLZF gene (shPLZF), SIRT1 (shSIRT1), or luciferase (shCON) were both carried out using the shRNA design program of Genepharma website . The shRNAs were constructed into adenovirus plasmids, where the adenoviruses were generated based on procedures given in the reference . The seqvia the inferior vena cava was administered for the isolation of primary hepatocytes from C57BL/6J and Ko-SIRT1 mice. The viability assessment of hepatocytes was carried out using the trypan blue exclusion method. For the experiments, cells with viability >95% were used. For the culture of mouse hepatocytes, RPMI-1640, which contained FBS (10%), penicillin (with concentration of 100 units/ml), and streptomycin (with concentration of 0.1 mg/ml), was used. In general, for overexpression experiments, we used 100 multiplicity of infection (MOI), and for shRNA knockdown experiments six00 MOI. Afterward, the harvesting of the cells was carried out after elapse of 1–2 days from the infection.The isolation of mouse primary hepatocytes and their culture was done following reference . In brieA commercial kit  was used for the purpose of determining the serum concentrations of TG and cholesterol following the manufacturer’s instructions. For determining the intracellular TG and cholesterol content, first cells were lysed with Triton X-100 (0.1%) and then a commercial kit  was employed for assessing the TG and cholesterol content of cell lysates. Hepatic TG and cholesterol were extracted with chloroform-methanol (2:1) according to reference. . and the® 96 Real-Time PCR System (Roche). Three independent biological replicates were carried out, and the genes’ relative expression levels were calculated with the 2−ΔΔCT method. The data of the gene expression were normalized to the expression levels of β-actin. The sequences of the primer can be found in The isolation of the RNA present in the mouse primary hepatocytes or fresh liver tissues was conducted by means of a TRIzol-based method (Invitrogen). Reverse transcription was performed according to the instructions of the manufacturer of the 5× All-In-One RT MasterMix kit . qRT-PCR was carried out utilizing SYBR Green Master mix with LightCyclerFor adenoviral transductions, HEK293T cells were transduced with adenovirus expressing Myc-tagged PLZF or Flag-tagged SIRT1. Once 36 h elapsed from transduction, whole cell lysates were retrieved by centrifuging . An amount of 1–1.2 mg of the lysates was utilized for the immunoprecipitation test. The incubation of lysates was carried out with anti-Flag antibody or anti-Myc antibody overnight (4 °C). Then, a 2 h treatment with protein A/G Sepharose was performed. After three-time rinsing with PBS, the process of retrieving the immunoprecipitated proteins from the beads was carried out through a 10 min boiling in sample buffer or by competition with the tag peptide. Afterward, immunoblotting was used for the analysis of immunoprecipitated proteins. Anti-FLAG mouse mAb  and Myc mouse mAb  were used against epitope tags.The lysis of cells and tissues was carried out in RIPA lysis buffer  plus a cocktail containing protease and phosphatase inhibitors (Roche), and another one comprising deacetylase inhibitors (Santa Cruz) for 30 min at 4°C. SDS-PAGE was used for the separation of an amount of 30–50 μg of proteins, which was transferred to PVDF membranes (Merck Millipore). Overnight incubation (4°C) with antibodies was applied to the membranes; the antibodies were the following: anti-SREBP-1 , anti-SIRT1 , PLZF , anti-FAS , anti-acetyl Lysine antibody  and β-Actin . For ac-PLZF examination, immunoprecipitation with an anti-PLZF antibody was introduced into cells and tissues extracts, which was followed by immunoblotting with an anti-acetyl-lysine antibody (α-AcK), so that only the acetylated PLZF protein was detected . DetectiFor H&E staining, neutral-buffered formalin (10%) was used for fixing the liver tissues; afterward, the tissues were paraffin-embedded, followed by being cut into sections (thickness = 7 μm) Liquid nitrogen was used for freezing the liver tissues for Oil red O staining; the frozen tissues were then cut into sections with a thickness of 10 µm. After staining the sections, their analysis was carried out at magnification of ×200 utilizing a Zeiss Axio Observer microscope.Primary mouse hepatocytes were seeded into six-well plates. In the next step, the indicated adenovirus was used for infecting them. Once 36 h elapsed from infection, the cells were washed with PBS (three times); afterward, they were incubated in 1 ml/well of phenol-red-free, glucose-free DMEM that contained dexamethasone (1 μM), pyruvate (2 mM), lactate (20 mM) and forskolin (10 μM) for 3–6 h. An Amplex Red Glucose/Glucose Oxidase Assay Kit  was used for determining the concentration of glucose in the medium. The lysis of the cells was performed, and for each lysate, the protein concentration was measured . The normalization of the glucose output rate was carried out using the cellular protein content.The co-transfection of mouse SREBP-1c promoter constructs  with PLZF-expressing plasmid (pcDNA3.1-PLZF) or empty vectors (pcDNA3.1) was carried out into HepG2 cells utilizing Lipofectamine™ 2000 (Invitrogen), in accordance with the instructions of the manufacturer. As an internal control, a vector expressing Renilla luciferase was utilized. After elapse of 48 h, the harvesting of the cells was performed to evaluate the luciferase activity utilizing the Dual-Luciferase reporter assay system (Promega), following the manufacturer’s instruction.3 (with concentration of 0.1 M) and SDS (with concentration of 1%)] and reversed. The amplification of promoter region of SREBP-1c was carried out by PCR utilizing these primers: 5′-GATTGGCCATGTGCGCTCA-3′ as a forward primer and 5′-CCTTCAAATGTGCAATCCATG -3′ as a reverse primer.ChIP assays were carried out using the procedure of a previous report . In briep < 0.05 was set as statistically significant.Data shown were presented as mean ± standard error of the mean (SEM). The software package IBM SPSS statistics (version 22.0) was employed for performing the statistical analysis. One-way ANOVA was utilized for the purpose of evaluating differences among groups followed by Bonferroni’s post hoc test. For making comparisons between two groups, differences were assessed by Student’s t-test. in vitro model of lipid accumulation, significantly upregulated PLZF expression in primary mouse hepatocytes  and injected Ad-shPLZF into NAFLD models, db/db mice with chow diet r weight . Moreover weight . Thus, ir weight . Importar weight , except DIO mice , which cin vitro experiments. According to the in vitro tests, consistent with previous reports (a (LXRα) (in vivo. Our results indicated that the promoter fragment of SREBP-1c (from +85 bp to −83 bp), which containing the E-box could be amplified while precipitating the DNA complex with anti-PLZF antibody treatment, but not when using normal mouse IgG (negative control) in the liver tissue lysate from C57BL/6J mice  . SREBP-1a (LXRα) . To detea (LXRα) , which s/6J mice . Finally/6J mice . These din vitro model of lipid accumulation, revealed that there is a remarkable interaction between PLZF and SIRT1  recruitment probably mediates the repressor-to-activator conversion . SIRT1 wnd SIRT1 . Consistnd SIRT1 . This phnd SIRT1 . Notablynd SIRT1 . Additiond SIRT1 .in vivo. According to our results, although the hepatic SIRT1 interruption did not diminish the increase in the body weight induced by overexpression of PLZF in the liver, the PLZF-induced increase in the ratio of liver weight to body weight in hepatic-SIRT1-deleted mice was significantly decreased  and HDACs for achieving repression; it also takes part in regulating a large number of target genes via zinc-finger-recognizable promoter elements  cascades. This phosphorylation was required for interferon-stimulated gene induction. Considering there is no commercial anti-phospho PLZF antibody is available at present, we have not detected the phosphorylation level of PLZF. However, in the future, further examination of the level of phosphorylated PLZF in the primary hepatocytes or tissues derived from mouse model of NAFLD could be detected by immunoprecipitated with a PLZF antibody firstly and then immunoblotted with anti-phospho Ser, anti-phospho Tyr antibodies. In addition, it was found that cyclin-dependent kinase CDK2 induced phosphorylation of another domain of PLZF, which resulted in impairment of the repression of the transcription; this indicated that phosphorylation could be counteracted by repression . In addition, with qPCR and western blotting analysis, we conformed that PLZF was relatively high expressed in liver and adipose tissues  in the PLZF coding sequence results in nonsynonymous amino acid substitution–serine to threonine at position 208 (T208S)–which affects total body weight, adiposity, and the insulin sensitivity of skeletal muscle . PLZF isde novo lipogenesis (DNL) due to being a carbon source for both fatty acids and glycerol synthesis, but also signally prompt upregulation of genes responsible for regulating fatty acid synthesis. The latter could be induced via direct and indirect processes: 1) in the direct scenario, the carbohydrate response element binding protein (chREBP) is essential (via mediation by carbohydrates (glucoses). The activation of ChREBP by glucose is via the regulation of the entry of ChREBP into the nucleus and by inducing the activation of transcription factor-DNA binding. Stimulation by glucose is required for binding of ChREBP to the promoter of liver-type pyruvate kinase (L-PK), which is a crucial enzyme that takes part in the regulation of glycolysis. The L-PK catalyzes phosphoenolpyruvate-to-pyruvate conversion, which steps into Krebs cycle to produce citrate, which is another source of acetyl-CoA utilized for the synthesis of fatty acids  ssential . ChREBP ty acids . Srebf1 ty acids . Herein,ty acids . Thus, avia directly binding to its promoter region. However, due to the dual-luciferase reporter assay was conducted in HepG2 cells which are cancer cells and might not really reflect the physiological activity of PLZF on SREBP-1c, therefore, a “normal” hepatocyte cell line, such as AML12 cell, maybe used to confirm this data in future. Furthermore, many studies have revealed the presence of an E-box in the promoter region of both PPARγ  almost abolished the PLZF regulatory effects. Thus, such data indicated that the effects of PLZF on SREBP-1c gene expression are through mediation by the E-box element. These findings suggest that PLZF promotes the transcription of SREBP-1c th PPARγ  and FAS th PPARγ . Howevervia binding to the promoter of SREBP-1c and upregulating lipogenic genes. Most importantly, we found that the requirement of hepatic SIRT1 for the dynamic switch from being a transcriptional repressor to being a transcriptional activator of PLZF depends on its deacetylase. This may present a potential therapeutic target for future attempts to treat NAFLD.In conclusion, we report on the identification of hepatic PLZF as a novel candidate gene for NAFLD


A Box–Behnkenexperimental design was implementedin modelwine (MW) to clarify the impact of copper, iron, and oxygen in thephoto-degradation of riboflavin (RF) and methionine (Met) by meansof response surface methodology (RSM). Analogous experiments wereundertaken in MW containing caffeic acid or catechin. The resultsevidenced the impact of copper, iron, and oxygen in the photo-inducedreaction between RF and Met. In particular, considering a number ofvolatile sulfur compounds (VSCs) that act as markers of light-strucktaste (LST), both transition metals can favor VSC formation, whichwas shown for the first time for iron. Oxygen in combination can alsoaffect the concentration of VSCs, and a lower content of VSCs wasrevealed in the presence of phenols, especially caffeic acid. Theperception of “cabbage” sensory character indicativeof LST can be related to the transition metals as well as to the differentphenols, with potentially strong prevention by phenolic acids. In this step,any oxygen present will be converted to its singlet form (simultaneouslyyielding ground state photosensitizer), with S1 oxygenbeing an electrophilic species responsible for nonradical reactionsinvolving compounds such as alkenes, sulfides, and amines (Type IImechanism).5 Alternatively, excited T1 RFundergoes reduction, acquiring two electrons from compounds such asthe amino acid methionine (Met) (Type I mechanism). The correspondingoxidation of Met yields methional, an unstable and light-sensitivemolecule, which produces methanethiol (MeSH) and acrolein througha retro-Michael reaction  having relatively low odor detectionthresholds in wine (2–10 μg/L for MeSH and 20–45μg/L for DMDS in white wine) and being responsible for odorsreminiscent of cooked cabbage, onion, and garlic  due to the reaction between hydrogen sulfide and thiols.11Upon further oxidation, two molecules of MeSH canyield dimethyldisulfide (DMDS),d in ref ). Furet 12 which is a concernwhen packaging wine in colorless (flint) glass bottles due to theextent of light transmission.13 The contentof RF in wine is around 150–200 μg/L and mainly relatedto the strain of Saccharomyces cerevisiae used for alcoholic fermentation,14 whereas thatof Met approaches 3–4 mg/L.17 Besides RF, wine containsother compounds sensitive to light, such as tartaric acid, whose photo-degradationproduces glyoxylic acid in the presence of iron,18 with subsequent browning phenomena occurring due to theformation of xanthylium cation pigments.19 Iron present in wine also catalyzes redox reactions and, along withcopper, is involved in the oxidation of phenols to quinones.20 Moreover, iron takes part in the formation ofStrecker aldehydes generated from the reaction between quinones andα-amino acids.21 To date, however,the effect of iron and copper on LST formation had apparently notbeen investigated.The appearance of unpleasantsulfur notes in wine can be associatedwith light-struck taste (LST),22 Copper is commonly used for the removal of “reduced”aroma defects from wine due to the formation of copper complexes withsulfhydryl compounds.22 However, thesecomplexes may behave as dissolved species and do not seem to be stableover time,23 meaning that copper is notable to definitively remove sulfur compounds associated with the “reduced”aroma defect. Moreover, copper addition to remove sulfhydryls is ineffectiveunder anoxic conditions.24 Indeed, to acertain extent, the presence of oxygen can limit the appearance ofdefects due to sulfur compounds or LST.25 Nonetheless, the proper management of oxygen is fundamental, especiallyfor white wine production, since oxidative phenomena involving phenolicsand the loss of desired aroma compounds, such as the varietal thiols,both need to be prevented.26Iron and copper in wine can participate inoxidative phenomenaat relatively low abundance , with averageconcentrations varying according to grape and wine production variables.Basedon the impact that iron, copper, and oxygen can have on oxidationand sulfide formation, the present study aimed to test the hypothesisthat the combined effect of iron, copper, and oxygen would influenceLST formation in a wine-like solution containing RF and Met. Thiswas investigated using a response surface methodology (RSM) approach,with the same experimental design being applied in the presence ofcatechin and caffeic acid as model phenols found in wine. Analysisof a number of oxidation products and VSCs was undertaken, along withan assessment of the sensory profiles of the samples. To the bestof our knowledge, this is first time that the combined effect of oxygen,iron, and copper on the formation of LST has been investigated. Themajor understanding of the conditions favoring the formation of unpleasantVSCs as a consequence of the light-mediated reactions between RF andMet allows the formation of an overall picture of complex photo-degradativemechanisms.o-phthalaldehyde (>99%) (OPA), l-Met (98%), methioninesulfoxide(Met sulfoxide) (98.5%), methionine sulfone (Met sulfone) (99%), d6-dimethyl sulfide (d6-DMS) (99%), isopropyl disulfide (96%), DMDS (98%), dimethyl trisulfide(DMTS) (98%), catechin (98%), caffeic acid (98%), sodium hydroxide(98%), and hydrochloric acid (37%) were purchased from Merck . All the chemicals were of analytical reagent grade, at aminimum. HPLC grade water was obtained from a Milli-Q system .Methanol (99.9%), ethanol (96%),acetonitrile (99.9%), RF (98%), citric acid (99.5%), tartaric acid(99.5%), copper sulfate pentahydrate (98%), iron sulfate heptahydrate(98%), magnesium sulfate heptahydrate (98%), 2-mercaptoethanol (99%), 27 Model wine (MW) solution samples were formulatedwith different levels of copper (as Cu2+), iron (as Fe2+), and oxygen, as outlined in 28 The samples that were protected from light during preparationand before and after controlled light exposure by covering with aluminumfoil were placed in hermetically sealed clear glass bottles (100 mL),and stored in the dark at 20 ± 2 °C until required. Thesamples were exposed to fluorescent light for 2 h using light bulbs(Philips) emitting cold light (6500 K) with a luminous flux of 4000lm, power of 65 W (26 × 8.8 cm), and high emission in the absorptionwavelengths of RF (370 and 440 nm). The illumination involved a specialtriangle-shaped apparatus of 40 cm per side with three lamps placedon the top, at a distance of 20 cm.30 An analogous experimental plan was implemented withsamples containing catechin (100 mg/L) or caffeic acid (70 mg/L),added from stock solutions prepared in methanol before the additionof the metals. Two samples were prepared for each treatment, withone being exposed to light and the other kept in the dark as a control.Experiments utilizing aBox–Behnken experimental design and RSM approach were set.1 Briefly,the samples were filtered through a 0.22 μm PVDF filter  and analyzed with an Acquity UPLC  system equipped with a photodiode array (PDA) detector recordingat 440 nm. Using an injection volume of 10 μL, separation wascarried out with a Hypersil ODS C18 column  maintainedat 25 °C with mobile phase A consisting of 90% (v/v) 50 mmolcitrate buffer at pH 2.5 and 10% methanol, and mobile phase B containing10% (v/v) 50 mmol citrate buffer at pH 2.5 and 90% methanol. The lineargradient increased from 0 to 70% B in 8 min at a flow rate of 0.6mL/min followed by column washing with 100% B for 3 min and columnconditioning with 0% B for 3 min. A six-point calibration curve wasobtained for RF concentrations prepared in MW in the range 20–500μg/L. Data acquisition and processing were performed with Empower2 software (Waters).The content of RF was assessedas described by Fracassetti et al.29 with some modifications. The derivatization solution was preparedin a 10 mL volumetric flask by dissolving 250 mg of OPA in 1.5 mLof ethanol, adding 200 μL of 2-mercaptoethanol, and making upto volume with borate buffer (0.4 M at pH 10.5). The derivatizationreaction was carried out at room temperature with 500 μL ofborate buffer, 200 μL of sample, and 100 μL of OPA solution.The reaction mixture was vortexed for about 1 min, filtered with a0.22 μm PVDF filter (Millipore), and injected after 5 min. Usingan injection volume of 20 μL, analysis was performed with anAquity UPLC coupled with a PDA detector recording at 338 nm. The columnwas a Waters Nova-Pak C18 . Mobile phase A was citrate buffer , and mobilephase B was acetonitrile/methanol/water (45/45/10 v/v/v). The lineargradient was 0–0.5 min, 5% B; 0.5–22 min, 47% B; and22–24 min, 100% B, followed by column washing with 100% for3 min and column equilibration with 5% B for 6 min. The flow ratewas set to 1 mL/min, and the column temperature was 40 °C. Six-pointcalibration curves were obtained in the range 0.1–10 mg/L foreach analyte in MW derivatized according to the method. Data acquisitionand processing were performed with Empower 2 software (Waters).Met, Metsulfoxide, and Met sulfone concentrations were determined after derivatizationwith OPA as described by Fracassetti et al.31 with piperidine solution  and sodiumnitroferricyanide solution  using a seven-point calibrationcurve (0–200 mg/L) of acetaldehyde in MW. The detection limit(LOD) of the method was 0.3 mg/L.Acetaldehyde concentrationwas determined with a colorimetric assay according to the OIV protocol30 Briefly, 5 mL of sample, 1.25g of magnesium sulfate heptahydrate, 5 μL of isopropyl disulfidesolution (12.5 mg/L in MW), and 20 μL of d6-DMS solution (125 mg/L in MW) were added to an SPME vialthat was immediately capped and stored in the dark until the momentof analysis. Isopropyl disulfide was used as an internal standardto quantify DMDS and DMTS, and d6-DMSwas used for MeSH. SPME was carried out with a 1 cm carboxen-polydimethylsiloxane-divinylbenzenefiber  using a HTA autosampler fitted to an Autosystem XL gas chromatograph coupledwith a Turbomass mass spectrometer . Theseparation was achieved with a Stabilwax-MS column  using heliumas carrier gas at 1 mL/min. Electron ionization mass spectra (70 eV)were recorded in scan mode at m/z 33–350. Duplicate injections were carried out for each sample.Results for free MeSH are expressed relative to the concentrationof d6-DMS (μg/L), whereas six-pointcalibration curves were prepared for DMDS and DMTS (0.5–200μg/L). The estimated total VSCs corresponds to the sum (in moles)of the sulfur compounds detected. The estimated ratio between themoles of sulfur compounds formed  and themoles of sulfur lost as degraded Met and formed Met sulfox was determined.25 The odor activity values (OAVs) were determinedas the estimated ratio between the amount of the VSC found in thesample and the respective perception threshold: MeSH, 0.3 μg/L;DMDS, 20–45 μg/L; and DMTS, 0.1 μg/L.8Free VSCs were analyzed by solid-phasemicroextraction (SPME)-GC/MS as reported by Fracassetti et al.32 carried out the olfactoryscoring for “cooked cabbage” descriptor using a nine-pointscale, with nine being the highest intensity (extremely perceived).30 MW spiked with Met (3 mg/L) and two different levels of RF (200or 400 μg/L) and exposed to light for 4 h was used to trainthe panelists so they were confident about the perception of cookedcabbage note.30 The judges were calibrated byolfactory assessment of metal-free and phenol-free MW spiked withMet (3 mg/L) and RF (200 μg/L) exposed to light for increasinglengths of time up to 2 h, with the latter being considered to havea score of 5. The samples were presented to the panelists in a randomizedorder under ambient temperature and light. Coded ISO glasses containing25 mL of sample and covered with a glass Petri dish were presentedto the panelists.Sensory evaluationwas conducted oneach trial for single samples of light-exposed and control MW samples.The panel consisting of six expert judges R2). Partial least squares (PLS) wasused to determine the regression models and establish the effectsproduced by the considered factors .33 The experimental variability was taken intoaccount by means of the three replicated experimental runs executedin the center of the experimental domain. These replicates were thenused in the ANOVA tests to evaluate and compare experimental and modelvariances. The significance of coefficients of factors  and their interactions , as well as the presenceof bias, were evaluated by means of ANOVA; only significant modelsare shown. Relationships between factors and predicted responses werethen evaluated through the analysis of regression coefficients. Contourplots for response surface analysis were prepared considering thepairs of factors  with the highesteffect and/or interactions. The third factor that was not representedin the plot was considered at the average concentration .Box–Behnkenexperimentaloutputs were analyzed using MODDE 6.0 (Umetrics). For the RSM approach,the fit of the model was evaluated by the coefficient of determination(One-wayANOVA with post hoc Fisher’s LSD (α = 0.05) was carriedout to determine the significant differences related to sensory analysis,using SPSS Win 12.0 program .34Photo-degradation reactionsof RF and Met were evaluated to investigateLST phenomena in the presence of iron, copper, and oxygen. A Box–Behnkenexperimental design and the application of RSM were used for treatmentsinvolving different concentrations of iron , copper, and oxygen . This designwas chosen to evaluate the possible impact of the three selected parameterson their own as well as their interactions. Moreover, the use of anRSM approach allowed for regression modeling (via PLS). Such modelscan be used to predict the behavior of the selected parameters providinginformation on how they can be managed in an oenological perspective.MW was adopted to avoid interferences from a multitude of matrix componentspresent in wine and more accurately follow the light-induced reactionsof RF and Met. The impact of caffeic acid and catechin, consideredas model phenols for white wine, was also considered in the light-inducedreactions between RF and Met. Phenols can act as antioxidants dueto their hydroxyl groups leading to low redox potential and, consequently,increased oxygen consumption.25 Met sulfone was not detected in any of the samplesirrespective of light exposure, in accord with a previous study.29 Met sulfoxide, which can be produced from light-inducedreactions in the presence of RF and Met,35 was detected in minor concentrations (0.10–0.15 mg/L) inruns 3, 4, 6, 10, 13, 14, and 15 (Table S1 of the Supporting Information). Evidently, Met sulfoxide formationmay be limited by a greater presence of oxygen  and favored in particularby iron at lower oxygen concentration, suggesting the major impactof the metal-catalyzed oxidation in comparison to oxygen-mediatedoxidation. In any case, Met sulfoxide cannot explain the entire decreasein Met29 as other unknown compounds canbe formed as a consequence of Met oxidation.36 The disappearance of Met could be explained by building PLS regressionmodels calibrated on each modeled response, including DMDS concentration,sum of VSC concentrations, estimated molar ratio of sulfur compoundformed/Met degraded, and cabbage sensory score. Models for these parameterswere found to be significant on the basis of ANOVA and were associatedwith the following R2: DMDS concentration,72.6%; total VSCs formed, 79.0%; Met lost, 79.6%; molar ratio of sulfurformed/Met degraded, 67.6%; and cabbage sensory score, 77.0%. R2, 17 and Fracassettiet al.25 reported a major decrease of Metunder anoxic condition as Type I photo-degradation can only occurwith Met acting as an electron donor. Moreover, the combination ofcopper and oxic condition could lead to the oxidation of other compounds that are more easily oxidizable than Met, andconsequently, lower consumption of Met can occur. However, lower degradationof Met does not necessarily mean that lower concentrations of VSCsare formed, as presented later.Exposingsamples to light in these experiments caused the complete photo-degradationof RF such that it was not detected (data not shown), whereas samplesmaintained in the dark had an RF concentration of 203 ± 7 μg/L. The concentration of Metin the samples kept in the dark was 2.96 ± 0.59 mg/L , with greater decreases in all the light-exposedsamples. The degradation of Met was predominant under anoxic conditionsand in the presence of transition metals, corresponding to an averagedecrease of about 30% 1. This w37 and was reported by Clark et al.18 in MW in the presence of tartaric acid and iron.The highest concentration of acetaldehyde was detected for the runwhere oxic condition was applied in the presence of iron .Acetaldehyde was determined because it can originatefrom ethanoloxidation by hydroxyl radicals induced by the Fenton reactionTable S2 of theSupporting Information) and it does seem to be partly affected bythe concentration of dissolved oxygen . The presence of both copper and iron affectedthe formation of free MeSH, and between the two transition metals,iron appeared to have a major impact (Tables S3 and S4) where copper was added, and relatively high values werealso found when Cu and Fe were present .This result indicated that copper does not allow the removal of VSCsand favored the development of DMDS and DMTS under the experimentalconditions adopted, in accord with previous work.38 The formation of DMDS has been recently suggested to involvea dimer radical cation species9 that mightnot be bound by copper. DMTS could originate upon storage from theoxidation of methional and MeSH39 as wellas due to the formation of H2S.40 With regard to DMDS, the total VSCs, and the molar ratio of sulfurformed/Met degraded, these were statistically significantly correlatedonly with oxygen   3A.41 andcan produce Met sulfoxide,35 a stable compoundthat cannot be oxidized further, thereby limiting the formation ofsulfur compounds.36 The interaction ofcopper and oxygen was positively related to the sensory perceptionof LST. Indeed, the impact of copper appeared evident based on whenthe concentration of this metal was the highest (0.5 mg/L), wherebyan increase of LST perception was observed even for the oxic condition(oxygen at 8 mg/L)  3B. MoreoTable S5 of the Supporting Information). This result indicated the possibleprotective effect of caffeic acid toward Met degradation, possiblybecause caffeic acid can compete with Met in donating electrons toRF in a Type I mechanism as well as reacting with singlet oxygen ina Type II mechanism, as observed for gallic acid.35 Met sulfoxide was detected at up to 0.5 mg/L in the runsin oxic condition and where oxygen was 3 mg/L , suggesting the major influence of oxygen on its formationeven if a regression model could not be obtained with regard to Metsulfoxide formation. Trace amounts (<0.06 mg/L) of Met sulfonewere detected only under oxic condition (data not shown), althoughthis could be considered a negligible amount, being 10 times lowerthan Met sulfoxide.As found in the earlier trials, light exposure of solutionscontaining caffeic acid also led to the complete degradation of RF(data not shown) compared to 225 ± 12 μg/L RF determinedin the samples stored in the dark. In most of the cases, the degradationof Met may have been favored with light under anoxia 2 and in S5). Moreover, the highestformation of acetaldehyde was detected in run 7 (7.21 mg/L) underoxic condition and in the presence of 5 mg/L iron. As previously mentioned,the increase of acetaldehyde can be related to the Fenton reaction.18Acetaldehyde was detected in all the runs but primarilyfavoredwith light exposure, as observed by an increase under most of theconditions tested. Higher concentrations were determined in the runswhere iron was present 2 and S5.35 and potentiallycounteracting the release of VSCs. Moreover, the oxidized form ofcaffeic acid could also bind MeSH, limiting its oxidation to DMDSand the formation of DMTS. With regard to the polysulfides, it seemsthat copper alone could favor the formation of DMTS, given the highestconcentrations were found in runs 2 and 9 . As found in MW, the temporary effect of copper bindingof VSCs42 may have also been evidenced,since free MeSH was detected even in the runs where copper was presentat the highest level considered in this study (0.5 mg/L). Nonetheless,comparing runs 5 and 6, the relative concentration of free MeSH waslower in the latter (−78%), which differed by the additionof copper (0.5 mg/L). Iron played an important role in the formationof this VSC even at 5 mg/L (the average level investigated) , producing quinones.These unstable quinones, specifically those deriving from the oxidationof caffeic acid, can react with the α-amino acids to generateStrecker aldehydes, with the corresponding reduction of quinones backto phenols. Oxygen was modeled as a negative contributor to free MeSHconcentration , those for DMDS were up to 1  (Table S3 ofthe Supporting Information), and those for DMTS were up to 1 (Table S4 of the Supporting Information), withthe exception of runs 2 and 9 in the latter case, where the OAVs weregreater than 10. Even so, the values for DMTS across the runs weremuch lower than those found for MW (Table S4 of the Supporting Information), thus supporting the important impactof phenolics against the formation of VSCs. Nonetheless, the modelswere not significant in the presence of caffeic acid, suggesting itspossible ability to prevent the appearance of LST.Concerning the formation of VSCs, in general,the most abundantVSC in experiments containing caffeic acid was free MeSH, with theexception of run 2, where DMTS was the highest 2. Nonethtigated) 2. The reree MeSH 4A. Negatree MeSH 4B. This positive4B. As prntration 4B in agrour plot 5A: the did i.e., 1. The imid i.e., 2 in compid i.e., 1. CopperTable S6 of the SupportingInformation), albeit lower in concentration compared to the experimentswith caffeic acid. A negligible content of Met sulfone was found (<0.06mg/L), again indicating the minor formation of Met sulfone in comparisonto Met sulfoxide.Consistent with the trials in MWand in the presence of caffeicacid, no RF was found after light exposure in the system containingcatechin (data not shown). In contrast, the RF concentration in thesamples stored in the dark was 214 ± 9 μg/L. Anoxic conditionand an average level of oxygen (3 mg/L) caused a major decrease ofMet (compared to a concentration of 3.29 ± 0.29 mg/L for samplesmaintained in the dark). However, with an average Met degradationof about −15% 3, the prS6 of theSupporting Information).Similar to the trials with caffeic acid, acetaldehydewas detectedin all runs, including those kept in the dark. The highest concentrationof acetaldehyde was detected in the runs where iron was present .In contrast, DMDS showed OAVs between 0 and 1 in all runs (Table S3 of the Supporting Information). Therefore,free MeSH and DMTS were considered to be the VSCs having a greaterimpact on LST, even in the presence of catechin. The chemical natureof phenols could also play a role in the perception of LST; as inmost of the cases, OAV values were higher with catechin in comparisonto caffeic acid (Tables S2–S4 ofthe Supporting Information), but negligible differences in the cabbagesensory score were found.In most of the cases, the major roleplayed by oxygen in termsof VSC concentrations was again highlighted, with their formationbeing limited under oxic condition in the presence of catechin 3. The fiR2 = 76.3%) and the cabbage sensory score (R2 = 73.0%) (The RSM approach for MW added withcatechin enabled the developmentof regression models that explained free MeSH concentration (= 73.0%) 6A. The r= 73.0%) 6B. The l= 73.0%) 7A. This = 73.0%) 4B vs 6B.= 73.0%) 6C, suppo= 73.0%) 7B.36 Nonetheless, in most of the experiments, the degradation of Metwas related to the formation of DMDS and DMTS—when the decreaseof Met was greater than 0.6 mg/L, more than 10 μg/L DMDS andDMTS were found. On the contrary, these VSCs were lower than 10 μg/Lwhen degraded Met was less than 0.6 mg/L.In conclusion, transition metalssuch as iron andcopper are well-knowncatalysts of oxidation reactions and can favor the formation of VSCs.Oxygen can also take part in such reactions, affecting the concentrationof VSCs that represent the marker volatiles of the wine fault knownas LST. To the best of our knowledge, this is first time that thecombined effect of oxygen, iron, and copper on the formation of LSThas been investigated. We hypothesized that these variables couldinfluence LST formation in a wine-like solution containing RF andMet. The major purpose was to have an overall picture of the complexphoto-degradative mechanisms. In particular, with our study, the influenceof iron has been shown for the first time, maybe due to its involvementin Strecker degradation, as it can favor the formation of VSCs besidesbeing involved into the photo-Fenton reaction generating glyoxylicacid. Copper on the other hand is commonly used for the depletionof “reduced” aroma defects, but had only a limited impactin the present case; indeed, it may lead to an increase of VSCs underanoxic condition. Ultimately, different combinations of oxygen, iron,and copper were found to play a very important role in the developmentof LST since different concentrations of VSCs and cabbage sensoryscores were revealed depending on the experimental conditions adopted.This suggests that the presence of oxygen at bottling and the levelsof iron and copper should be taken into account along with the concentrationsof RF and Met. In particular with regard to Met, its chemical degradationdid not lead to the formation of the off-flavor as other compoundscould also be originated .The addition of phenolicsminimized the decrease of Met, probablydue to the ability of phenols to compete with Met, both in the TypeI pathway as electron donors to the triplet state RF and in the TypeII pathway where they compete with Met in the reaction with singletoxygen. In terms of sensory effects, however, the modeling showedthat phenols affected LST depending on their chemical nature. Underthe experimental conditions, lower concentrations of VSCs were inmost of the cases found when caffeic acid was added in comparisonto catechin, thus potentially limiting the appearance of LST.29 Nonetheless,we cannot exclude that the other compounds generated from side reactionmechanisms  and/or the interactionsamong them could have an impact on LST intensity perceived. This hypothesisrequires further investigation.Overall, besides the presence of RF and Met, the susceptibilityof a wine to develop LST appeared to be related to the presence oftransition metals as well as to the different phenols that would ordinarilybe present in wine. It may be that wines with a higher content ofphenolic acids or phenols that are less easily oxidizable than flavan-3-olscould be less susceptible to the appearance of LST, but this aspectrequires further verification. VSCs determined in this study havebeen reported to be the main compounds responsible for the appearanceof LST.


Gallic acid (GA) and lecithin showed important roles in antioxidant and drug delivery, respectively. A complex synthesized from GA and soybean lecithin (SL-GAC), significantly improved bioavailability of GA and pharmacological activities. However, the antioxidant activity of SL-GAC and its effect on iron-overload-induced liver injury remains unexplored.in vitro and in mice, and its remediating effects against liver injury by iron-overloaded.This study investigates the antioxidant properties of SL-GAC In vitro, free radical scavenging activity, lipid peroxidation inhibition, and ferric reducing power of SL-GAC were measured by absorbance photometry. In vivo, C57BL/6J mice were randomized into 4 groups: control, iron-overloaded, iron-overloaded + deferoxamine, and iron-overloaded + SL-GAC. Treatments with deferoxamine  and SL-GAC  were given to the desired groups for 12 weeks, daily. Iron levels, oxidative stress, and biochemical parameters were determined by histopathological examination and molecular biological techniques.In vitro, SL-GAC showed DPPH and ABTS free radicals scavenging activity with IC50 values equal to 24.92 and 128.36 μg/mL, respectively. In C57BL/6J mice, SL-GAC significantly reduced the levels of serum iron (22.82%), liver iron (50.29%), aspartate transaminase (25.97%), alanine transaminase (38.07%), gamma glutamyl transferase (42.11%), malondialdehyde (19.82%), total cholesterol (45.96%), triglyceride (34.90%), ferritin light chain (18.51%) and transferrin receptor (27.39%), while up-regulated the levels of superoxide dismutase (24.69%), and glutathione (11.91%).in vivo and in vitro studies are needed to validate its potential in clinical medicine.These findings encourage the use of SL-GAC to treat liver injury induced by iron-overloaded. Further  Reactive oxygen species (ROS) are typical by-products of cellular metabolism, playing a role as secondary messengers and influencing different normal physiological functions of the body , a naturally occurring phenolic acid widely distributed in a variety of foods such as apples, b7lueberries, walnuts, and watercress, is well known as powerful antioxidant  was obtained from Novartis International AG . Carbonyl iron, GA, soybean lecithin, ethanol, vitamin C, ferric chloride, ferrous chloride, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2-azinobis (3-ethylbenzo-thiozoline-6-sulfonic acid) (ABTS), potassium ferricyanide and 30% hydrogen peroxide were purchased from Sigma Chemical Co. . The assay kits for alanine transaminase (ALT) (C009-2-1), aspartate transaminase (AST) (C010-2-1), γ-glutamyl transferase (γ-GT) (C017-2-1), and superoxide dismutase (SOD) (A001-3-2), glutathione (GSH) (A006-2-1), malondialdehyde (MDA) (A003-1-1), total cholesterol (TC) (A111-1-1), total iron-binding capacity (TIBC) (A040-1-1), triglyceride (TG) (A110-1-1) were procured from Nanjing Jiancheng Biotechnology Co., Ltd. . Antibodies against transferrin receptor (TfR1) (ab84036), Ferritin (ab153976), ferritin light chain (FL) (ab109373) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab198306) were all purchased from Abcam Inc. .The SL-GAC, a stable complex of soybean lecithin and gallic acid was prepared according to our previously described method  was produced by reacting ABTS•+ solution with 140 mM potassium persulfate and allowing the mixture to stand in the dark for 12–16 h at room temperature before use. Subsequently, the ABTS•+ solution was diluted in ethanol to obtain an absorbance of approximately 0.51 ± 0.02 at 734 nm. A volume of 0.6 mL of SL-GAC with different concentrations was added to 2.4 mL amount of ABTS•+ solution for 10 s, and stood for 6 min in the dark. The reference compound (vitamin C) was used at the same concentration as the SL-GAC. Thereafter, the absorbance of all test tubes was determined at 734 nm. The solution containing only anhydrous ethanol served as the blank and all determinations were carried out in triplicate. The percentage of inhibition of ABTS•+ was calculated using the following formula:0 = absorbance of the blank sample, and A1 = absorbance of the test sample.The ABTS radical cation scavenging activity was determined according to the method of Re et al. . ABTS waThe ferric thiocyanate method  and incubated at 37 °C for 1 h. The reaction mixture without sample was used as control. The hydroxyl radical was investigated by monitoring absorbance at 536 nm.Different concentrations of the SL-GAC solution  were added to reagent solution  to the ferrous ion (Fe2+) by antioxidants in the sample  assay. This method is based on the reduction of the ferric ion , model + DFO (FED), and SL-GAC groups, with each group containing 10 mice. Except for the control group, which was provided with a regular diet and physiological saline, the other three groups were fed with ordinary diet supplemented with 3% carbonyl iron for 12 weeks to induce iron-overload condition. In the meantime, from week 2, in addition to the same treatment as that of the FE group, mice of FED and SL-GAC groups were intraperitoneally injected with DFO (150 mg/kg body weight), intragastrically administered with SL-GAC (200 mg/kg body weight) every day, respectively. DFO is poorly absorbed from the gastrointestinal tract when taken orally, resulting in a decrease of its bioavailability. Therefore, iron overload-induced liver injury mice were treated with DFO via the method of intraperitoneal injection. Moreover, SL-GAC ameliorated significantly the hepatic damage and iron overload in a mouse ALD model induced by alcohol in our previous study, and the mice were intragastrically administered with SL-GAC. In the present study, we conducted experiment with reference to the administration of SL-GAC on mice ALD model, and the selected administration were effective on iron overload-induced liver injury mice model.The animal study was performed according to the guidelines established by the National Institutes of Health, and with permission from the Animal Experiment Committee of Jilin University . Healthy male C57BL/6J mice (8 weeks old) were purchased from Changsheng Experimental Animal Company , and were kept at 23 ± 2 °C under a 12 h dark/light cycle, with a relative humidity of 50–55% and allowed access to normal laboratory pellet diet and water At the end of experiment of the study, and after 12 h of food deprivation, the excrement of mice was collected and frozen in liquid nitrogen. Then, all mice were narcotized with lidocaine and killed by cervical dislocation, the blood was collected from each of the mice by cardiac puncture for biochemical analysis. After the clotting of blood samples, serum was separated by centrifugation and stored at −80 °C until analysis. Liver was immediately excised, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C for further analyses. One part of the liver tissues was fixed in 4% neutral formalin for histopathological analysis.g and stored at −80 °C until used for biochemical analysis. The level of ALT, AST and γ-GT was measured by automatic biochemical analyzer according to the manufacturer’s instructions. Serum iron, liver iron and TIBC were measured with the iron and iron binding–capacity kit. Serum iron concentration was determined using the assay based on the generation of an iron-ferrozine complex. Iron concentration in the digested liver sample was measured spectrophotometrically at 535 nm, following reaction with 2 mM bathophenanthroline disulfonic acid. The TS was directly calculated from the serum iron and TIBC, and UIBC was equivalent to the sum of TIBC and serum iron.Serum samples of mice were prepared by centrifuging the whole blood for 8 min at 2,000 Liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as possible. Then, liver was weighed and homogenized at a ratio of 1:9 (w/v) in 0.9% saline solution. The homogenates were centrifuged at 3,000 rpm for 10 min at 4 °C, the supernatant was harvested and the levels of MDA, GSH, SOD, TG and TC were determined using commercial kits according to the manufacturer’s instructions.The smaller liver fragments (3 × 3 × 3 mm) were embedded by optimal cutting temperature embedding agent in the pre cooled cryostat. After frozen, the tissue sections were made, the thickness was about 5 mm, and 5 M dihydroethidium (be configured with PBS) was used at 37 °C. After incubation for 15 min, rinsed slices with PBS for 3 times and observed under confocal laser scanning microscope . Fluorescence intensity was observed, each group of three samples, the same exposure intensity and time to take photographs.Mice liver tissue samples were removed from −80 °C and lysed in radioimmunoprecipitation (RIPA) lysis buffer, followed by grinding with tissue lyser at 60 HZ for 2 min at 4 °C and then put on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 rpm at 4 °C. Bicinchoninic acid kit for determination of the protein concentrations of collected supernatant. Next, equivalent amounts of protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk for at least 2 h and then incubated with primary antibody at 4 °C overnight, followed by incubation with a horseradish-peroxidase-conjugated secondary antibody at room temperature for 2 h. The protein bands were visualized using enhanced chemoluminescence. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was used as a control.Body weights and liver tissues of mice were measured at the termination of the study. Hepatic index was estimated from the ratio of liver tissue weight to body weight (liver tissue weight/body weight × 100).Liver specimens from central zone of medial lobe were cut into about 3 mm thick, fixed in 10% neutral buffered formalin for 12 h and then embedded in paraffin. Next, the paraffin block was sliced into 5 μM thick sections, stained with hematoxylin-eosin according to the manufacturer’s instructions, and examined microscopically for histopathological changes. Meantime, immunohistochemistry (IHC) assay for ferritin of sections obtained from 10% paraffin-embedded tissues was performed by the one step polymer-HRP Detection Kit (BioGenex) according to the manufacturer’s protocols. Subsequently, these stained sections were visualized and photographed under inverted microscope (100 × magnification).t-test and one-way analysis of variance. p < 0.05 was considered to indicate a statistically significant difference. Calculations were performed with the SPSS, Version 26, statistical software package.All data were presented as means ± SEM. Differences among groups were assessed by using Student’s in vitro, DPPH, ABTS and hydroxyl radical scavenging activities, linoleic acid autoxidation inhibition capacity and Fe3+ reduction capacity were used. As shown in 50 values of 24.92 ± 0.46 µg/mL and 128.36 ± 6.57 µg/mL, respectively compared to vitamin C (IC50 = 44.78 ± 0.62 μg/mL and 114.30 ± 0.61 µg/mL). In addition, scavenging activity of SL-GAC on Fenton hydroxyl radicals increased with increase in the concentration of SL-GAC. Excitedly, the ability of SL-GAC to scavenge hydroxyl radicals in Fenton reaction were stronger than those of vitamin C at the same concentrations (3+ showed an accelerating trend with increased concentrations of SL-GAC (in vitro.For the determination of antioxidant capacity of SL-GAC trations . Moreovetrations . Furtherf SL-GAC . The resp < 0.01) (p < 0.01)  . Additio < 0.01) . But TIB < 0.01) . These rFerritin is a hepatic protein that plays vital roles in diagnosing and predicting diseases, its levels reflect body iron stores and are elevated with inflammation in chronic liver injury , interestingly, treatment with SL-GAC reversed the increase of serum ALT, AST and γ-GT that led to reduced liver injury induced by iron overload in mice (p < 0.01)  .Oxidative stress is a major pathogenetic event occurring in several liver disorders ranging from metabolic to proliferative ones. The effects of SL-GAC on ROS levels in liver tissue of iron-overloaded mice were detected using the Dihydroethidium staining. As shown in p < 0.01) (p < 0.01). These results demonstrated that SL-GAC can effectively attenuate lipid peroxidation and reduce oxidative stress induced by iron-overload in mice.The levels of SOD, GSH, and lipid peroxidation product MDA were evaluated to assess the level of oxidative damage and the antioxidant effects of SL-GAC. As expected, the iron overloaded mice showed increased MDA while SOD and GSH levels decreased compared with blank mice. Conversely, the model mice supplemented with SL-GAC showed markedly reduced MDA with elevated SOD and GSH levels in comparison to the FE group ( < 0.01) . We furtvia HE staining revealed that the structure of hepatic lobules was intact in the blank group, as characterized by the radiating arrangement of hepatic cells and cords around the central vein, as well as the hepatic sinusoids were regularly distributed. In FE group, the hepatic lobule structure appeared disintegrated, meanwhile, proliferating fibrous tissues and pseudolobules were seen (black arrows), and scattered lymphocyte infiltration (green arrows) in the liver interstitium were observed in the liver tissues. Photomicrographs of the liver in the SL-GAC group showed a relatively normal hepatic architecture, with a preserved parenchymal structure and occasional infiltration of inflammatory cells (green arrows) (p < 0.01), and while that of FED and SL-GAC groups showed a significant recovery compared with model group (p < 0.01)  , indicat arrows) . Along w < 0.01) . These rIron plays a crucial role in the body, it is important for us to maintain an adequate supply of iron to synthesize oxygen transport proteins, and participates in numerous processes necessary for normal body functions Wallace . As ther3+) and ferrous (Fe2+) states. Because iron converts easily between its ferrous (Fe2+) and ferric state (Fe3+), many proteins utilize iron as a cofactor to catalyze redox reactions  can participate in the Fenton or Fenton‐like reactions to react with H2O2 and generate one of the most deleterious reactive oxygen species, hydroxyl radical; see reaction (1)  Zhao .(1)Fe2+in vivo, and susceptible to lipid peroxidation mediated by oxygen free radicals. Currently, lipid peroxidation is considered as the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that leads to cell death  binds to TfR1, Fe2-Tf is internalized by endocytosis  and aspartate aminotransferase (AST) are released into the extracellular compartment, which increases levels of ALT and AST in serum and further reflects the degree of hepatocyte injury to a certain extent (Goorden et al. via the Fenton reaction, which causes oxidative damage and even triggers cell death, ultimately causing hepatic injury (Dixon and Stockwell Excessive iron increases ROS production Liver index is an important indicator to measure hepatic injury in animal experiments, and it has been used to evaluate the degree of experimental liver damage (Zhu et al. in vitro studies concluded that the SL-GAC possesses promising antioxidant capacity and free radical scavenging properties. Further, in vivo studies reported the ameliorating effect of SL-GAC against iron-overload-induced liver damage. Treatment with SL-GAC reduces the accumulation of excess iron in serum and liver tissue, restored serum parameters, restrains ROS production and reduces oxidative stress in liver tissue. Further, hepatoprotective effect of SL-GAC were confirmed by physiological indexes and histopathological analyses (Accumulation of excess iron causes oxidative damage of biomolecules, which causes tissue injury primarily liver dysfunction. Observations from analyses . Collect