Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per mL sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities. Aspergillus, Fusarium, and Penicillium species are the most detrimental ones because these so-called aflatoxins, ochratoxins, trichothecenes , and zearalenone have various adverse effects on the health of humans and animals [Aspergillus species, has the strongest adverse effects on health, as it might lead to liver cancer [−1) in grain, corn, nuts, and fruits, as published in (EG) Nr. 1881/2006. Especially due to those harsh effects and corresponding low regulatory limits, highly sensitive and reliable detection technologies are of primary importance.According to the Food and Agriculture Organization of the United Nations and a recent study by Eskola et al. in 2019, approximately 25% of food-crops worldwide are contaminated with mycotoxins, a group of secondary metabolites produced by molds [ animals ,5,6,7,8.r cancer ,11,12. SCurrently, there are basically three analytical technologies used for mycotoxin testing. On the one hand, laboratory-based liquid chromatography (LC) coupled to mass spectrometry (MS) is the most common method, as well as enzyme-linked immunosorbent assay (ELISA), are routinely used for highly sensitive mycotoxin testing. On the other hand, lateral flow assays (LFA) are used for fast but less sensitive on-field testing ,17,18,19−1  up to 50 µg·mL−1, for example, Ridascreen Aflatoxin B1 30/15 test kit  or AgraQuant Aflatoxin B1 ELISA test . Nevertheless, due to the requirement for laboratory equipment, their usability for on-site testing is very limited. In such cases, commercially available LFA have the highest potential for fast and cost-effective on-site mycotoxin testing. However, most of these assay systems provide only qualitative results with LODs ranging from 4 ppb to 5 ppb, as achieved with RIDA®Quick Aflatoxin test kit or AFB1 (Aflatoxin B1) lateral flow assay kit , respectively. Primarily due to matrix interference effects, these assays can result in false-positive detection [In the field of ELISA, the most commonly used format for aflatoxin detection is the competitive assay because of the small molecular structure of the antigen that prevents the simultaneous binding of two antibodies, which would be the prerequisite for a sandwich ELISA. The basic working principle is the coating of mycotoxin-conjugate onto a microtiter plate followed by the addition and incubation of a mycotoxin-specific antibody in the presence of a liquid extract of sample material. In this step, mycotoxins in the sample compete with coated mycotoxins for antibody-binding. Subsequently, antibodies saturated with soluble mycotoxins are removed during a washing step and cannot contribute to a signal, achieved by either direct readout or indirect readout by means of a labeled secondary antibody. In this format, a low signal corresponds to a high concentration of mycotoxin within the sample, and vice versa. Most of the currently commercially available ELISA-based assay formats have a limit of detection (LOD) ranging from 1 µg·mLetection ,23.A novel, portable magnetic immunodetection approach has been described in previous studies ,26,27,282) has an amplitude of a few millitesla, resulting in alternating positive and negative magnetic saturation of superparamagnetic particles oscillating with a frequency of 2f2 of 122 Hz [1) with 49 kHz probes the magnetization state of the superparamagnetic particles and yields an iron oxide dose-dependent signal when the low-frequency driving field is close to zero. Finally, the resulting mixing frequency signal of f1 + 2f2 can be demodulated and detected by the innermost coil, composed of two adjacent sections, so-called detection coil (upper one) and reference coil (lower one). Those sections differ only in the winding-orientation of coils. With this clock- and counterclockwise orientation yielding induced voltages of opposite sign, the directly induced excitation field can be canceled out. By placing the sample carrying the MPs in the detection head, the resulting signal is amplified, measured, and directly visualized at the touchscreen of the handheld, portable magnetic reader  and FMMD. The sensitivity should be comparable to a laboratory-based ELISA. Hence, initially, a competitive ELISA (cELISA) was established, serving as a reference method. Assay parameters, such as the used coating and antibody concentrations, were optimized to reach a sufficient sensitivity for the detection of aflatoxin B1. Afterward, the cMID assay was established using the same optimization strategy in combination with further evaluation of the required amount of nanoparticles. The basic principle of cMID is the use of biotinylated antibodies, which can be enriched within the coated immunofiltration column by a competitive binding reaction depending on the amount of pre-captured mycotoxin. By flushing magnetic particles functionalized with streptavidin through the column by gravity flow, particles can bind to retained antibodies and subsequently be detected using FMMD. −1 to 5000 ng·mL−1 sample buffer. Absorbance was measured at 405 nm by indirect readout using a secondary antibody conjugated to horseradish peroxidase (HRPO) targeting mouse antibodies and application of respective substrate.As a reference method to novel cMID, a cELISA was established using optimized conditions defined by checkerboard titration. Here, aflatoxin B1-BSA conjugate was coated, followed by the application of aflatoxin B1-specific monoclonal antibodies pre-incubated with soluble aflatoxin B1 with concentrations ranging from 0.006 ng·mL−1 up to 5 µg·mL−1 in combination with aflatoxin B1-specific monoclonal antibody (mAb) AFB1_002 concentrations ranging from 19.5 ng·mL−1 up to 1250 ng·mL−1 were tested in a 96-well plate. Increasing signals were achieved with antibody concentrations above 19.5 ng·mL−1 and coating concentrations up to 0.6 µg·mL−1. Coating of AFB1-BSA with higher amounts than 0.6 µg·mL−1 resulted in saturated readout signals at all antibody concentrations that did not further increase. Considering sensitivity, the lowest possible amount of antibody should be used with coating concentrations resulting in highest possible signals. As a consequence, combinations of 75 ng·mL−1 or 150 ng·mL−1 antibody with 0.2 µg·mL−1 and 0.4 µg·mL−1 coating of AFB1, respectively, seem to be best suited for further experiments.In order to determine optimal aflatoxin B1-BSA (AFB1-BSA) coating concentrations and the appropriate amount of aflatoxin B1-specific monoclonal antibody to obtain the highest possible sensitivity, a checkerboard titration test was performed. As shown in −1 to 50,000 ng·mL−1. The combination of 0.2 µg·mL−1 coating and 150 ng·mL−1 antibody resulted in the highest sensitivity in combination with stable data behavior. Corresponding IC50 and Limit of Detection (LOD) values of all four combinatorial experiments are shown in −1 should theoretically lead to a higher sensitivity, this was actually not observed due to divergent measuring values and thus a non-reliable assay procedure under these conditions (compare 0.4 µg·mL−1 coating and 75 ng·mL−1 mAb). Furthermore, reducing the amount of mAb resulted in a twofold increase of readout time from approximately 10 min to more than 20 min (data not shown).To identify the optimal assay parameters, calibration experiments were performed with the previously most promising combinations of AFB1-BSA coating and aflatoxin B1-specific monoclonal antibody AFB1_002 concentrations . Free af50 value of 3.79 ng·mL−1 and an averaged LOD of 0.39 ng·mL−1, as shown in Considering the reliability of the developed cELISA, a further repetition of the best-paired concentration setting, as shown in −1 up to 10 µg·mL−1 with one column per condition. Remaining binding sites were blocked with a BSA solution. Then aflatoxin B1-specific biotinylated monoclonal antibody AFB1_002 in concentrations ranging from 0.3 µg·mL−1 up to 10 µg·mL−1 were applied onto these columns after pre-incubation with 180 µg·mL−1 of 700 nm superparamagnetic streptavidin-functionalized particles  have a maximum binding capacity of 600 ng·cm2, whereas ABICAP immunofiltration columns can bind up to 24,000 ng·cm2, which in fact explains the absence of signal saturation at the used conditions. A further difference when using checkerboard titration for assay optimization of cELISA or cMID can be seen in fluctuating readout signals, whereas in ELISA, concentration-dependent signals were obtained with almost no variability between consecutive measuring points. In contrast, signals generated in MID checkerboard titration varied, especially at high antibody concentrations. This could be caused by an overdose of magnetic particles, which might result in sterical hindrances and a partly erratic aggregation of particles within the assay matrix. Based on this hypothesis, a magnetic-particle dose adaptation was made by reducing the applied concentration towards robustly detectable saturated measuring signals  since the molar concentration is proportional to the molecular weight of the particles. By applying a higher ratio of small particles onto the column, a higher amount of small MPs could bind to antigens coated on the column surface. Another explanation could be the occurrence of steric hindrance within the ABICAP immunofiltration matrix when using large particles. Especially if there is a low concentration of free mycotoxin within the sample, a high amount of biotinylated antibody will be retained within the matrix in a competitive assay approach, resulting in a high antibody density on the matrix surface. When large MPs are applied, steric hindrance can occur at the surface. Either multiple, closely located biotinylated antibodies could be linked to one MP, or multiple antibodies could be blocked due to the size of large MPs. Here, especially the thousand-fold increased volume of 700 nm diameter MPs in comparison to 70 nm MPs in diameter might have the most important role. By the greatly increased size and volume, several antibodies are covered by one single MP, so that they are no longer accessible for binding of further MPs. By using smaller particles, shielding effects and sterical hindrances at the surface are reduced, resulting in a higher magnetic particle density within the column, and finally, a higher readout signal. A similar effect observed by Achtsnicht et al. (2019) when using small 75 nm instead of large 1010 nm particles in their MID experiments was also explained by the blocking of remaining binding sites [−1 was found when using the large particles, but a more than 15-fold lower LOD of 0.2 ng·mL−1 could be reached using the small-sized MPs. In our study, especially due to the high standard deviations at high measuring signals when exciting the 700 nm MPs for aflatoxin B1 detection, a reliable calculation of LOD or IC50 values was not possible. However, by using 70 nm particles, high and stable readout signals in combination with a perfectly matching nonlinear-fit (R2 = 0.9938) enabled the reliable calculation of LOD and IC50 values of 1.1 ng·mL−1 and 5.4 ng·mL−1, respectively, which are in the same sensitivity range as our comparative lab-based cELISAHowever, the resulting cMID assay did not lead to the expected results when using 700 nm magnetic beads. Switching to 70 nm magnetic particles instead resulted in a tenfold increase of measuring signal combined with reduced variability and provides a basis for a highly sensitive and reliable assay . The resng sites . The autAlthough the best performance of the cMID assay was achieved with 70 nm small magnetic particles, the general applicability of 700 nm large particles in cMID assays should not be completely neglected since, in contrast to small particles, larger particles can be separated in a gradient magnetic field. However, the possibility of magnetic separation and thereby enrichment of magnetic particles might play a crucial role in sample preparation where cleanup and concentrating of an analyte is required to achieve sufficient assay sensitivity, as it was used for example by Lee et al. (2013) or Xuan and colleagues (2019) ,31. In tThe 70 nm particles used in this study cannot be efficiently separated in a magnetic gradient field, since their magnetic attraction is low, which was shown by Achtsnicht and colleagues (2018) . They co−1. Based on our findings, it can be concluded that the competitive magnetic immunodetection is a powerful tool for portable, easy-to-use on-site monitoring of mycotoxin contamination in various matrices to reduce the risk of processing contaminated food and agricultural products. In future work, we will focus on the further optimization of the cMID procedure using the 70 nm MPs. Especially, a significant reduction of the assay time from currently approximately 4.5 h to less than one hour should be addressed, similar to that described by Rettcher et al. (2015) [In conclusion, we successfully demonstrated the development and implementation of a competitive magnetic immunodetection assay for the detection and quantification of aflatoxin B1 with a LOD of 1.1 ng·mL. (2015) . There a. (2015) . By usin. (2015) . Here th2HPO4 × 12 H20, KH2PO4, Na2(CO3), NaHCO3, Milk powder, and Albumin Fraction V (biotin-free) were acquired from Carl Roth, Karlsruhe, Germany.Dimethyl sulfoxide, Tween-20, Aflatoxin B1, Aflatoxin B1-BSA, EZ-Link™ NHS-PEG4 Biotinylation Kit, ABTS buffer, as well as ABTS tablets were purchased from Merck KGaA, Darmstadt, Germany. NaCl, KCl, Na2CO3 and 35 mM NaHCO3 in MilliQ-water, and pH was set to 9.6 with glacial acetic acid. Phosphate buffered saline (PBS) was prepared by dissolving 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 × 12 H2O, and 1.5 mM KH2PO4 in MilliQ-water and setting pH to 7.4 with hydrochloric acid. As a washing buffer, PBS-T was produced by adding 0.05% (v/v) Tween-20 to PBS. ELISA blocking buffer (EBP) was prepared by adding 5% (w/v) milk powder to PBS. For magnetic immunodetection experiments, blocking solution consists of 1% (w/v) albumin fraction V (biotin free) in PBS, and is called MID-BP. All other chemicals, except Tween-20, were acquired from Roth.The used coupling buffer was prepared by dissolving 15 mM Na®-CLD/synomag®-CLD; article number 05-19-502 S09718) as well as 70 nm streptavidin-functionalized magnetic particles  were purchased from micromod Partikeltechnologie GmbH, Rostock, Germany.Immunofiltration columns (ABICAP HP columns) were purchased from Senova Gesellschaft für Biowissenschaft und Technik mbH, Weimar, Germany. High binding 96-well microtiter plates (article number 655061) were purchased from Greiner Bio-One GmbH, Frickenhausen, Germany. Anti-mycotoxin monoclonal antibody AFB1_002 was purchased from fzmb GmbH, Bad Langensalza, Germany. Secondary antibody goat anti-mouse IgG-HRPO (article number 115-035-008) was purchased from Jackson ImmunoResearch Europe Ltd. UK. 700 nm streptavidin-functionalized magnetic particles  and incubated as mentioned above. After washing each well thrice with PBS-T, all wells were blocked using 200 µL of EBP and incubated. Then, the plate was washed again, and concentrations of monoclonal antibody ranging from 19.5 ng·mL−1 ABTS substrate in ABTS buffer was added, and absorbance was measured at 405 nm after 15 min incubation in the dark.For competitive ELISA procedure, AFB1-BSA conjugate was diluted in coupling buffer and plated with 100 µL per well onto a high binding 96-well microtiter plate. As mentioned in −1 was applied, as well as for cMID assays. Subsequent washing of the columns was performed twice with 750 µL PBS. Remaining binding sites were blocked by applying twice 750 µL of MID-BP. After the first 750 µL flushed through the column by gravity flow, an incubation time of 5 min was set. After the second time, columns were incubated for further 30 min after they were washed again twice with PBS.The equilibration of immunofiltration columns was done, as described by Rettcher et al. (2015) . In brieAfter equilibration or blocking, columns can be stored in the coupling buffer or PBS, respectively, at 4 °C for at least 14 days. In this study, a maximum storage time of one day was used.For biotinylation of antibodies, the EZ-Link™ NHS-PEG4 Biotinylation Kit was used as described by the manufacturer’s instruction.−1 and 10 µg·mL−1, were applied and incubated for one hour at room temperature. Afterward, a washing step was performed by rinsing two times 750 µL PBS through the column. Subsequently, for checkerboard titration, 500 µL of 180 µg·mL−1 700 nm magnetic beads suspension in PBS (pH 7.4) was added and flushed through by gravity flow. For bead response analysis, various concentrations of 700 nm magnetic beads were applied. Another washing step, as described above, was performed. For readout, columns were inserted into the portable magnetic reader and the measuring signal in mV was detected, as previously described in Rettcher et al. (2015) [After coating and blocking of immunofiltration columns, 500 µL samples of biotinylated antibody, diluted in PBS to various final concentrations between 0.3 µg·mL. (2015) .−1 to 100,000 ng·mL−1, were mixed 1:1 with 225 µL biotinylated antibody, also diluted in PBS. After incubation of one hour at room temperature in a dark surrounding, 500 µL of each sample was applied on coated and blocked columns and also incubated as mentioned above. After washing each column twice with 750 µL PBS, 80 µg·mL−1 of 70 nm or 700 nm magnetic particles were applied and incubated, as mentioned before. After washing twice, the readout was done as described above.For cMID calibration curve experiments, a pre-incubation of free mycotoxin and biotinylated antibody was performed. For this, serially diluted aflatoxin B1 samples in PBS, with concentrations ranging from 0.006 ng·mLFor competitive ELISA as well as for competitive magnetic immunodetection and data analysis, a Hill Slope fit was done with GraphPad Prism 8.0.0. The following formulas were used to determine the LOD on the signal and on the concentration scale:


All formulations showed microparticles, neutral pH, and negative zeta potential (–6 to –27 mV). They were non-irritating and nontoxic and showed high permeation through bovine sclera. Formulations containing carboxymethyl cellulose (CMC) showed greater anti-inflammatory activity, even higher than the commercial formulation, Nevanac® 0.3%. The optimized formulations represent an opportunity for topical instillation of drugs to the posterior segment of the eye.The aim of this study was to design and evaluate novel cyclodextrin (CD)-based aggregate formulations to efficiently deliver nepafenac topically to the eye structure, to treat inflammation and increase nepafenac levels in the posterior segment, thus attenuating the response of inflammatory mediators. The physicochemical properties of nine aggregate formulations containing nepafenac/γ-CD/hydroxypropyl-β (HPβ)-CD complexes as well as their rheological properties, mucoadhesion, ocular irritancy, corneal and scleral permeability, and anti-inflammatory activity were investigated in detail. The results were compared with a commercially available nepafenac suspension, Nevanac Inflammation of the eye and surrounding tissues is among the ocular pathologies with the highest incidence in ophthalmology, which, deprived of the appropriate treatment, can lead to visual loss . The mai®, in two doses, 1 mg/mL three times per day and 3 mg/mL once per day [® 0.1% three times a day, as they both showed the same efficacy.Treating inflammation at the posterior segment classically involves ophthalmic steroids as well as nonsteroidal anti-inflammatory drugs (NSAIDs) ,9. The r per day . The sid per day . Sahu et per day  analyzed per day  demonstrUnlike other NSAIDs, nepafenac is a prodrug. After topical ocular treatment, it penetrates the cornea and is transformed by ocular tissue hydrolases into its active metabolite, amfenac , an inhi®, Voltaren®, Vitaseptol®, and Indocid®) [Treating the posterior segment in eye diseases by means of topical instillation is still a challenge, due to the different biological membranes and physical boundaries of the eye that restrict drug passage and penetration ,24,25. Nndocid®) . Combinandocid®) ,39,40. Rndocid®) , and the® 3 mg/mL suspension. To the best of our knowledge, this is the first time that formulations containing γ-CD/HPβ-CD nanoparticles have been evaluated in vitro and ex vivo.This study was aimed at the development and evaluation of novel CD-based aqueous eye drop formulations containing mucoadhesive polymers  and comparing their effectiveness with previous formulations developed to efficiently deliver nepafenac to the eye in order to treat inflammation, increase drug concentration in the posterior segment, and reduce the expression of inflammatory mediators. All aggregate formulations were evaluated for in vitro diffusion studies, rheological and mucoadhesive properties, in vitro anti-inflammatory activity, and ex vivo corneal and scleral permeability studies. They were compared with a commercial NevanacNepafenac  was acquired from Fagron ; γ-cyclodextrin (γ-CD) was provided by Wacker Chemie ; 2-hydroxypropyl-β-cyclodextrin, DS 0.62  was kindly donated by Janssen Pharmaceutica . Methyl cellulose  was from ICN Biomedicals Inc. ; sodium alginate  was from Fagron Iberica ; sodium hyaluronate  was from Guinama .®, Madrid, Spain). All other reagents were analytical grade.Benzalkonium chloride (BAK), ethylenediamine-tetraacetic acid disodium salt dihydrate (EDTA), reagent-grade tyloxapol (MW 280.4 g/mol), 87%–90% hydrolyzed poly (PVA) , hydroxypropyl methylcellulose , polyvinylpyrrolidone , and carboxymethylcellulose (CMC) sodium salt  were purchased from Sigma-Aldrich . Membrane filters (0.45 µm) were obtained from Phenomenex . Water was purified using reverse osmosis  and buffer solution B .Carbonate buffer, pH 7.2, was prepared by mixing buffer solution A  and THP-1 monocytes (ATCC TIB-202™) were purchased from the American Type Culture Collection . Fetal bovine serum (FBS), antibiotic solution , lipopolysaccharides (LPS) from E. coli, phorbol 12-myristate 13-acetate (PMA), tris hydrochloride, and lauryl sulfate sodium salt (SDS) were acquired from Sigma-Aldrich . Dulbecco’s Modified Eagle’s Medium with Ham’s F-12 Nutrient Mixture (DMEM/F12) and RPMI 1640 were supplied by Gibco . WST-1 Cell Proliferation Reagent was purchased from La Roche .Nepafenac (18 mg) was added to 6 mL of an aqueous 15%γ-CD/8%HP-βCD (w/v) solution containing different polymers , 0.1%  and centrifuged at 4000 rpm for 15 min at 25 °C , and supernatant was diluted with Milli-Q water. The apparent nepafenac solubility was determined by UV-Vis spectroscopy at 254 nm using a standard calibration curve previously validated in triplicate in the range 3–25 µg/mL.Subsequently, suspensions were placed in an ultrasonic water bath  at 60 °C for 60 min. They were cooled down to room temperature and kept in a shaker  under constant agitation for 7 days at 37 °C. After this, suspensions were filtered (AcrodiscParticle size and size distribution of formulations A1 to A9 and Nevanac was measured by dynamic light scattering (DLS) using a Nanotrac Wave particle analyzer . Samples that were previously filtered were diluted with Milli-Q water, and measurements were carried out at 25 °C with a 780 nm laser and 180° scattering angle. Each measurement was done in triplicate.® 3000HS. pH was measured with a GLP22 pH meter . All measurements were done in triplicate at 25 °C.Zeta potential of formulations A1 to A9 was recorded using a Zetasizer–1. Data analysis was carried out using Rheology Advantage data analysis software. Experiments were performed using 1.5 mL for each formulation.Rheological characterization of formulations was carried out using a Rheolyst AR-1000N rheometer  equipped with an AR2500 data analyzer, a Peltier plate, and a cone . First, storage (G′) and loss (G″) moduli were recorded at 37 °C and 0.1 Pa applying angular frequency sweeps from 0.1 to 50 rad/s. Viscosity and flow curves were performed under rotational runs at 37 °C for 2 min with shear stress in the range 0.1 to 200 sMucoadhesion strength was evaluated in triplicate using a TA.XT Plus Texture analyzer  following methods previously described by Akhter et al.  and CampThe ocular irritation test was carried out as previously reported . Briefly® CCL-163TM) murine fibroblasts using the WST-1 test. BALB 3T3 cells were cultured in DMEM/F12 culture medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Gibco). They were seeded in a 96-well plate at 1.5 × 104 cells/well. To allow complete cell attachment, cells were incubated 4 h at 37 °C and 5% CO2. Aliquots of A1 to A9 formulations, Nevanac 3 mg/mL suspension, and control (DMEM/F12) were diluted 1:50, 1:100, and 1:150 times, respectively, with complete cell culture medium to be below the IC50 of nepafenac, to ensure that nepafenac was not in cytotoxic concentrations, and added to cell monolayers [The cytocompatibility of cyclodextrin formulations was evaluated on BALB/3T3 clone A31 (ATCCnolayers . DMEM/F12. Samples (1 mL) were taken from the receptor phase at 30, 60, 90, 120, 180, 210, 240, 300, and 360 min and replaced with fresh medium. Commercial eye drops, Nevanac 3 mg/mL, were also tested. Nepafenac content was determined by UV-VIS spectrophotometry at 254 nm using a method previously validated with standard solutions in the range of 3–25 µg/mL.Nepafenac diffusion tests from eye drop formulations were performed in triplicate in vertical Franz diffusion cells fitted with cellulose acetate membrane filters . Membrane filters were soaked in the receptor medium for 1 hour before starting the experiment. The donor phase consisted of aliquots of 1.00 mL of the test formulation. The receptor phase was 6.00 mL of 2.5% (w/v) γ-CD/HPβ-CD ratio (80/20) aqueous medium to ensure sink conditions, and kept at 37 °C and under magnetic stirring at 300 rpm. The diffusion area was 0.786 cm2), C0 is the initial concentration of nepafenac in the formulation (g/mL), and D is the diffusion coefficient (cm2/min).Diffusion coefficients (D) were estimated from the Higuchi equation:2 area available for permeation). Samples (1 mL) were removed from the receptor chamber at 0.5, 1, 2, 3, 4, 5, and 6 h, replacing the same volume with carbonate buffer each time, and taking care to remove bubbles from the diffusion cells. All experiments were carried out in triplicate.Ex vivo corneal and scleral permeability studies of selected nepafenac formulations were carried out using fresh bovine eyes from a local slaughterhouse. The eyes were kept in phosphate-buffered saline (PBS) solution with antibiotics previously added (penicillin 100 IU/mL and streptomycin 100 μg/mL) and maintained in an ice bath during transport. Corneas and scleras were isolated, washed with PBS, and placed on vertical diffusion Franz cells. Both receptor and donor phases were filled with carbonate buffer, pH 7.2, following the bovine corneal opacity and permeability (BCOP) protocol, placed in a bath at 37 °C, and kept under magnetic stirring for 1 h in order to balance ocular tissues. After this, the buffer in the donor chamber was completely removed and replaced by the formulations (2 mL). Chambers were covered with parafilm to prevent evaporation , with a C18 column  and ChromNAV software. The mobile phase consisted of acetonitrile: water (50:50) at a flow rate of 1 mL/min and 90 µL for injection volume and retention time of 1.9 min.® syringe filter, 0.22 µm GHP Minispike, Waters) into small Eppendorfs, centrifuged again , and filtered to be measured by HPLC.After a 6 h permeation test, aliquots of the donor chambers were taken for HPLC analysis. Previously injected corneas and scleras were excised and nepafenac content was extracted in tubes with 3 mL of ethanol/water (50:50 v/v) mixture for 24 h at 37 °C, and sonication was applied for 90 min in an ultrasound bath at 37 °C. Afterwards, tubes were centrifuged , and the supernatant was filtered (Acrodiscapp) was calculated from the flux (J) according to Equation (3):0 is the initial concentration of nepafenac in the donor phase. Each experiment was performed in triplicate and the results are reported as mean values ± standard deviation (SD).The apparent permeability coefficient (PTHP-1 human monocytes (ATCC TIB-202™) were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Hyclone), 2-mercaptoethanol , and 1% penicillin-streptomycin (Gibco). Phorbol 12-myristate 13-acetate  200 nM was used to promote the differentiation of THP-1 monocytes into macrophages . Previou4 cells/well. To induce an inflammatory response, macrophages were treated with 100 ng/mL of lipopolysaccharides (LPS) from Escherichia coli O111:B4  and incubated at the same time for 24 h at 37 °C and 5% CO2 with the samples. Cells treated with only LPS served as positive controls, while unstimulated cells (without LPS) were used as negative controls. Formulations A2, A3, A5, A8, and A9 were selected for anti-inflammatory efficacy, and their corresponding blank formulations (without the drug) were also tested as controls.After macrophage differentiation, PMA solution was removed and cell monolayers were washed with Dulbecco’s phosphate buffered saline (DPBS) and trypsinized following standard protocols. Cells were seeded into 48-well plates at 4.5 × 10After incubation, cell culture supernatants were collected and stored at –150 °C until cytokine assessment. The secretion of 3 inflammatory mediators, PEG-2, IL-6, and IL-1ra, was analyzed. The concentration of PEG-2 was studied using an EIA assay (Arbor Assays), while IL-6 and IL-1ra were analyzed by specific ELISAs  after adequate dilution following the manufacturer’s instructions.p < 0.05.Data are presented as mean ± standard deviation (SD). The effect of formulation composition on anti-inflammatory response was analyzed using ANOVA and multiple range test . Differences were considered significant at The apparent solubility, zeta potential, and pH of the designed formulations of nepafenac are summarized in In the formation of ternary complexes, drug-CD-polymer has been widely explored to enhance solubility and dissolution of poorly soluble drugs ,48. One All formulations showed negative zeta potential, in good agreement with the anionic or nonionic nature of the polymers and surfactants involved. Formulation A6 showed the highest absolute value of zeta potential, –27.4 ± 1.7 mV. Although a high absolute value of zeta potential is related to the high stability of nanoaggregates, other factors such as ionic strength, pH, or amount of encapsulated drug can influence aggregation behavior .Moreover, all pH values were about 6.08–6.21, i.e., in a range adequate for ocular administration . ParticlAll formulations tested presented microparticles (approx.5–6 µm), and formulations A3, A5, and A9, which contained PVA and CMC or PVP and CMC, displayed also small portion of smaller particles (less than 1 µm). Compared to the particle size of Nevanac reported by Shelley et al. , the incShort precorneal residence time limits the ocular bioavailability of drugs formulated as conventional topical eye drops. One strategy to prolong precorneal residence relies on the addition of polymers that can increase viscosity and therefore drug retention in the ocular sac . Viscosi–1), and low viscosity during blinking , which is appropriate for eye drop formulations [It is known that upon application of mechanical force such as eye blinking, viscosity of the natural tear film decreases in a pronounced manner. Eye drops should ideally display pseudoplastic behavior to prevent their removal under blinking conditions . As showulations . RegardiViscoelastic behavior at 37 °C was also analyzed , recordiRegarding viscoelastic behavior, adding 1% CMC, 0.2% HA, and 0.4% SA to aggregate formulations containing nepafenac/γ-CD/HP-βCD modified the rheological properties of the formulations . FormulaIt is known that after topical instillation to the eye, precorneal factors and the reflex mechanism of the eye lead to rapid drug elimination, and only a small fraction of the drug is available at the ocular surface. Treatment of eye diseases with mucoadhesive delivery systems has been proposed as a strategy to enhance the drug retention of topical ophthalmic formulations. Polymer–mucin bonds can be used to trap formulations on the surface of the eye, thus increasing the thickness of the tear film . SeveralNevanac displayed the highest mucoadhesive strength (0.672 ± 0.03N), followed by formulation A4, which contained CMC and HA; A2, which contained CMC; and A5, with PVP, HPMC, and CMC.Cellulose derivatives such as CMC and HPMC and sodium hyaluronate have been extensively used as mucoadhesive polymers. Brako and co-workers  studied As mucoadhesion is correlated with viscosity , these rThe Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) test is based on the detection of vascular damage in the chorioallantoic membrane, which is an analog for ocular conjunctiva . DiffereThe percentage of cell survival relative to the negative control of fibroblasts treated with formulations A1 to A9 and Nevanac at three dilutions is shown in Nevanac was diluted with DMEM/F12 medium to obtain final concentrations of nepafenac of 0.236 mM, 0.118 mM, and 0.0787 mM, since EC50 was reported to be 0.0875 mM . All forAll samples tested were shown to not be harmful to BALB 3T3 cells, with cell viability similar to that exhibited by the marketed formulation, Nevanac. Results confirmed that a dilution of 1:100 was adequate for further assessment of anti-inflammatory activity.Nepafenac diffusion from aggregate formulations and marketed suspension was first evaluated in vitro under sink conditions for six hours. A cellulose acetate membrane  was used to separate the donor from the receptor compartments .2), followed by formulation A5 (847.8 ± 39.5 µg/cm2), A2 (808.9 ± 31.6 µg/cm2), and A3 (749.6 ± 58.4 µg/cm2). The increased diffusion compared to Nevanac is due to a higher fraction of solubilized nepafenac. Since diffusion depends on concentration gradient and formulations A2, A3, A5, and A9 are the ones that showed the greatest solubilizing capacity of the drug.Formulation A9 showed the fastest diffusion , which can be attributed to the high viscosity of the formulation at the ocular surface. On the other hand, in sclera higher accumulation was found for formulations A9 (87.76 ± 8.08 µg/cm2), A8 (65.84 ± 6.34 µg/cm2), A5 (71.07 ± 7.64 µg/cm2), and A2 (79.64 ± 1.90 µg/cm2). These differences between corneal and scleral accumulation are probably due to aggregate formation. Furthermore, consistent with some reports, sclera demonstrated higher permeability compared to cornea [After six hours, Nevanac accumulated the most onto the surface and inside the bovine cornea  receptor antagonist (IL-1ra), among other anti-inflammatory molecules, at the ocular surface . Alternap < 0.05; n = 3) was observed for all formulations compared to the positive control. In the case of IL-6 ( of IL-6 B, all fo of IL-6 C, NevanaSeveral studies have measured the concentration of inflammatory mediators after treatment with anti-inflammatory drugs to assess their therapeutic activity. Kern et al.  analyzed® 3 mg/mL. This provides an alternative for the topical delivery of hydrophobic drugs such as nepafenac used to treat ocular diseases at the back of the eye. In the meantime, further studies should be conducted to assess its feasibility in vivo.In summary, we successfully developed cyclodextrin-based aggregate formulations capable of delivering nepafenac to the posterior segment of the eye via the sclera to treat inflammation. All suspensions were found to be nonirritating and biocompatible after HET-CAM assay and in vitro cell viability assay in murine fibroblasts. The optimal eye drop formulation, A9, containing CMC, PVA, and MC, showed high drug solubilizing capacity, high sclera retention, and a higher reduction of the inflammatory response compared to a marketed formulation, Nevanac


The aim of this study was to investigate HIF-1α, HIF-2α, and ProExC expression in conjunctival intraepithelial neoplasia (CIN), to differentiate between metaplasia and dysplasia, and to access their value as diagnostic and prognostic immunohistochemical markers. Recurrence and progression into SCC (squamous cell carcinoma) were defined as endpoints.Forty-three specimens including CIN I (2), CIN II (9), CIN III (29), with and without metaplasia, and metaplasia alone (3), as well as 21 conjunctival control specimens, were stained with antibodies against HIF-1α, HIF-2α, and ProExC. The percentage of positively stained cells were calculated and used for further analysis.p < 0.001). Upper epithelial cells in CIN were more often ProExC-positive compared with normal conjunctiva or metaplasia (p = 0.06 and p = 0.07). Cox proportional-hazards analysis was performed for characterization of factors influencing the combined endpoint and showed a significant elevated hazard ratio for staining with ProExC (p = 0.04) compared with HIF-1α (p = 0.26) and HIF-2α (p = 0.49).The mean percentages of HIF-1α and HIF-2α were not increased in CIN. In comparison, the expressions of these markers were even significantly elevated in control specimens (Our study shows that HIF-1α and HIF-2α do not serve as diagnostic or prognostic markers in CIN. ProExC seems to be a potential indicator for CIN, but not a reliable diagnostic marker. However, control specimens occasionally also display a high percentage of ProExC-positive cells and staining over the entire epithelial layer. Conjunctival intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC) are rare tumors but are among the most common conjunctival malignancies with an incidence of SCC between 0.03/100000 persons per year in the USA and 3.4 and 3.0 cases/year/100000 in Zimbabwe .CIN can present as a simple dysplasia to carcinoma in situ. The diagnosis of dysplasia and the grading of CIN are made histopathologically by means of subjective assessment. Classification and grading parallel cervical intraepithelial neoplasia . CIN grade depends on the proportional distribution of dysplasia in relation to the epithelial height . By analDysplasia is defined by morphological criteria such as atypical, enlarged, and/or hyperchromatic nuclei; increased mitosis; and apoptosis. These criteria are in some extent quite subjective. Therefore, additional immunohistochemical stains supporting malignancy would be helpful, also in differentiating those tumors consisting of metaplasia alone from those with CIN and additional metaplasia.In cervical IN, the markers HIF-1α and ProExC have already been evaluated as diagnostic markers –7.A higher expression of HIF (hypoxia-inducible factors) was linked to poorer prognosis in a large number of different tumors –9. HIFs,In cervical IN, studies observed a higher expression of HIF-1α in high-grade cervical IN compared with that in normal cervical epithelium , 7. FurtProExC consists of two monoclonal antibodies against TOP2A (topoisomerase 2α) and MCM2 (minichromosome maintenance protein 2) which are two important factors in DNA replication , 15. ProDue to the similarity between cervical and conjunctival intraepithelial neoplasia, we focused on the markers HIF-1α and ProExC as potential diagnostic and prognostic markers of CIN. Besides this, we aimed to investigate HIF-2α in CIN which level of expression has been found increased in various tumors like bladder, breast, colon, and also cervical carcinomas .The study was approved by the ethics committee of the Albert-Ludwig University Freiburg, Germany; written informed consent was obtained from the study participants. Forty-three conjunctival specimens with diagnosed CIN or metaplasia (details in results) which had been excised at the Eye Center Freiburg between 2004 and 2013 were included. Control specimens originated from excess conjunctiva that had been resected at the end of buckle retinal detachment surgeries.After excision, the specimens were fixed in 4% formaldehyde in phosphate buffer of 7.0 pH for 24–48 h, dehydrated in increasing concentrations of ethanol (70–100%) and xylol and infiltrated with paraffin . Sections of 4 μm thickness were cut and floated on deionized water at 20 °C, and single sections were mounted on X-tra adhesive glass slides . Slides were stretched in a water bath at 47–48 °C, subsequently dried at 60 °C for 30 min, and stored at a room temperature in darkness.Prior to immunohistochemical staining, the specimens were deparaffinized and rehydrated by xylol and in decreasing concentrations of ethanol (100–70%). Afterward, they were demasked using a citrate buffer of pH 6, heated in deionized water at 95 °C for 20 min, and slowly cooled down for 20 min. Now, they were stained immunohistochemically using the catalyzed reporter deposition method by biotin-free tyramide amplification with the DAKO CSA II kit and washed with 0.05 M TBST-wash buffer between the steps as recently described . First, Two to five representative sections of each specimen were photographed at a magnification of × 400 (HIF) and × 200 (ProExC), respectively. The cell number was determined by a self-programmed, validated, automated, cell counting software (reliability of 100%). Positive nuclear staining for HIF-1α and HIF-2α, and a strong cytoplasmic staining for HIF-2α were determined by counting manually, whereas positively stained cells for ProExC were counted automatically by the computer-based calculator. The percentage of positively stained cells for HIF-1α, HIF-2α, and ProExC was calculated. The results were subdivided into three groups: strongly (> 1.10% (HIF-1α), > 9.38 % (HIF-2α), and > 59.19% (ProExC) positively stained cells), medium (0.43–1.10% (HIF-1α), 1.27–9.38% (HIF-2α), and 43.5–59.19% (ProExC) positively stained cells), and weakly (< 0.43% (HIF-1α), < 1.27% (HIF-2α), and < 43.5% (ProExC) positively stained cells) expressed. In addition, the distribution of positively stained cells for ProExC regarding the vertical extension of epithelial thickness was determined and divided into basal, middle, and upper third.To investigate whether inflammation might have an impact on the immunoreaction, we also counted manually all intraepithelial as well as subepithelial inflammatory cells (lymphocytes) in each photograph. Four groups were defined as 0: no inflammatory cell, 1: < 5 inflammatory cells, 2: 5–20 inflammatory cells, and 3: > 20 inflammatory cells.We also reviewed the medical records for the following: age at time of surgery, sex, histopathological diagnosis, and days until tumor-associated events .p value < 0.05.We fitted a proportional hazards Cox regression model in order to assess the influence of the aforementioned covariates on tumor-related events. Significant level was set at The study cohort comprised 12 women and 31 men with a median age of 63 years (27–87 years) at time of surgery. Of these 43 included specimens, 3 were diagnosed with metaplasia alone, 8 with CIN without metaplasia, and 32 with CIN and metaplasia. Referring to the CIN specimen, 2 were classified as CIN I, 9 as CIN II, and 29 as CIN III. The median follow-up period was 1013 days . None of the included patients developed metastases or died in association with the included disease. Recurrence was shown in 9 patients (23%) after a median time of 301 days , most of them within the first 2 years. One was initially diagnosed with CIN I, 2 with CIN II, and 6 with CIN III. Three patients (7%) developed SCC after 441, 806, and 2653 days. All of them were male patients with initial diagnosis of CIN III and concomitant autoimmune dermatitis. In metaplasia, there was—as expected—no tumor-associated event. The control group comprised 21 normal conjunctival specimens from 9 female and 12 male patients with a median age of 59.5 years (33–82 years)   Fig. .Fig. 3HIp < 0.001)  Fig. .Fig. 4HIp = 0.55).The proportion of ProExC-positive cells in CIN was similar to the control group with a mean percentage of 50.6% ± 23.1% in CIN without metaplasia, 50.2% ± 20.9% in CIN with metaplasia, and 45.2% ± 17.3% in control specimens (R2 < 0.04).Our analysis showed that there was no correlation between extent of inflammation and immunostaining (p = 0.06).With regard to vertical extension of ProExC, we observed a correlation between the classification and the positively stained epithelial height, however missing statistical significance. About 52% of the normal conjunctival specimens showed positive staining for ProExC particularly in the basal third of the epithelium. In about 60% of the specimens with CIN, in contrast, cells in the upper third of the epithelium expressed ProExC (p < 0.001)  Fig. .Fig. 5PrNo statistically significant difference of HIF-1α and HIF-2α expression was found between CIN specimen developing recurrence or progressing to SCC compared with CIN without any of these tumor-related events. Notably, the mean expression of HIF-1α in specimens with CIN recurrence and progression to SCC were even lower (Table p = 0.12) and progression to SCC (p = 0.19) compared with those without any tumor-related event, but without statistical significance. Moreover, all three specimens with progression to SCC during the observation period were stained up to the upper third of the epithelium. Nevertheless, the difference of those cases without progression to SCC was not statistically significant (p = 0.34).In the Cox regression, we observed a higher ProExC expression in specimens with CIN recurrence  accumulate in tumors . We assuThe significantly increased expression of HIF in normal samples compared with CIN was unexpected, given the external oxygen supply. Our analysis hint towards intraoperative hypoxia in the controls 20–45 min by many severed capillaries before the control specimen was completely separated from the adjacent conjunctiva at the end of the surgery. The expression of HIF-1α level of brain tissue increased significantly under experimental conditions in rats within less than 1 h after traumatic head injury . For thiFurthermore, we investigated ProExC as a potential indicator for the classification of conjunctival specimens such as CIN and the course of the disease, but not as a reliable diagnostic marker. ProExC, a mix of two monoclonal antibodies against TOP2A and MCM2, has already been tested as a sufficient marker for proliferating cells and aberrant S-phase.p = 0.55). This is in contrast to the findings of Guo et al. who investigated cervical specimens and showed a staining in over 50% of the dysplastic cells in high-grade cervical IN [p = 0.04). Evaluation of the vertical extension of ProExC staining showed that upper epithelial cells were more frequently stained in CIN compared with normal conjunctiva or metaplasia, however missing statistical significance (p = 0.06 and p = 0.07). Furthermore, we observed a statistically significant correlation between the proportion of ProExC-positive tumor cells and the vertical extension (p < 0.001).In our study, no significant difference in the proportion of ProExC-positive cells in CIN compared with normal conjunctival specimen was found (vical IN . Neverthvical IN . RegardiA major drawback of our study is the low sample size. Our mostly negative findings may be due to a statistical power issue. Other limitations are the retrospective nature and the monocentric setting. Therefore, any clinic-related bias cannot be ruled out. However, a prospective large multicentric study is hard to perform because the conditions are very rare.In conclusion, neither HIF-1α nor HIF-2α seems to be suitable as a diagnostic or prognostic marker in CIN. ProExC may be of some value for predicting recurrences or progression to SCC when analyzing the proportional staining. These findings should be confirmed in patients from other eye centers with a larger sample size.


Liver receptor homolog 1 (LRH1) plays a vital role in several human cancers, but its role in ovarian cancer (OC) remains unclear. We aimed to explore the functions of LRH1 and its clinical relevance.in vitro. Furthermore, bioinformatics analysis was applied to predict the functions of LRH1.LRH1 expression was evaluated by immunohistochemistry and reverse transcription quantitative polymerase chain reaction (RT-qPCR). The effects of LRH1 on tumor cell proliferation, migration and epithelial–mesenchymal transition (EMT) were evaluated P < 0.05). LRH1 overexpression was extremely related with elevated International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.001), lymph node metastasis (P = 0.011), peritoneal metastasis (P = 0.001), and platinum resistance (P = 0.037). Furthermore, LRH1 expression was an independent prognostic index for disease-free survival in patients with OC (P = 0.041). LRH1 overexpression (P = 0.011), FIGO stage (P < 0.001), and ascites (P = 0.015) independently affected peritoneal metastasis in patients with OC. LRH1 knockdown significantly inhibited the proliferation, migration, and EMT of human OC cells (P < 0.05); however, it reversed cisplatin resistance. Bioinformatics analysis indicated that the functions of LRH1 were associated with the PRC1 complex, nuclear ubiquitin ligase complex, and Polycomb-group (PcG) proteins.RT-qPCR showed that LRH1 mRNA expression was higher in the invasive lesions (in vitro, and its functions may be associated with PRC1 complex, nuclear ubiquitin ligase complex, and PcG proteins.This study provides evidence of the predictive value of LRH1 on peritoneal metastasis and poor outcome and highlights the potential role of LRH1 as a biomarker for the targeted therapy of OC. Furthermore, LRH1 promotes OC cell proliferation, migration, and EMT  Ovarian cancer (OC) is the most lethal gynecologic malignancy among women. An estimate of 21,750 new cases of OC will be diagnosed, and 13,940 women will die of OC in the United States in 2020 according to the American Cancer Society . The proLiver receptor homolog 1 (LRH1), also known as nuclear receptor subfamily 5 group A member 2 NR5A2, participates in various biological processes, such as liver and pancreas differentiation, steroidogenesis in childhood, cholesterol/bile acid homeostasis, and tumor progression , 11. PreThus, we aimed to explore the status of LRH1 protein expression in OC and its clinical relevance. The functions of LRH1 were predicted using bioinformatics analysis.OC tumor tissue samples were obtained from Harbin Medical University Cancer Hospital between January 2009 and December 2011 from 133 patients with OC who underwent cytoreductive surgery without any prior treatment for cancer, such as chemotherapy or radiation. The International Federation of Gynecology and Obstetrics (FIGO) staging system was applied to assess tumor stage . HistoloApproval from the Medical Ethics Committee of Harbin Medical University Cancer Hospital was obtained for the purpose of research. The 133 patients with OC were followed up for survival analyses until death or until the study closing date (January 2018).133 tissue blocks of OC and 25 of normal ovary tissues were cut by a microtome into 4-µm sections and affixed onto the slide. After dewaxing in xylene and rehydrating through graded alcohol concentrations, the sections were incubated with 0.3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase, and then the sections were incubated with anti-LRH1 antibody  overnight at 4°C. After washing in phosphate-buffered saline (PBS), all sections were incubated with secondary antibodies  at room temperature for 20 min and then treated with 3,3′-diaminobenzidine tetrahydrochloride , followed by counterstaining with hematoxylin.Protein expression level of LRH1 was scored by evaluating the percentage of the positive staining areas of tumor cells together with intensity of staining. The former was scored as follows : 0, < 0%The IHC staining on each slide was scored twice independently by two pathologists who were sophisticated in evaluating IHC and blinded to the clinicopathological information.The human OC cell lines A2780CP, SKOV3 and OVCAR3 (ATCC) were employed. Both the cell lines have been approved by short tandem repeat (STR) profiling. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in DMEM complemented with 10% fetal bovine serum (FBS)  and antibiotics (penicillin and streptomycin). Cells were passaged when they reached 80% confluence.β-catenin ) at 4°C overnight and with secondary antibodies at 1:5,000 dilution at room temperature for 2 h, followed by incubating with β-actin  at room temperature for 1.5 h.The cell lysates were both centrifuged at 13,000 rpm (at 4°C for 5 min). After that 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) were used to segregate them and then transferred onto polyvinylidene difluoride (PVDF) membranes . The filters were blocked with blocking buffer (Sangon Biotech) for 45 min. Membranes were incubated with primary antibodies anti-LRH1 , anti-E-cadherin , anti-N-cadherin , anti-Vimentin , anti-® PCR System 9700 . Real-time PCR was performed using LightCycler® 480 II Real-time PCR Instrument . The mRNA expression levels of LRH-1 were normalized to the mRNA levels of GAPDH, which was used as an internal control. The 2−ΔΔCt method was used to quantify the mRNA expression levels (The TRIzol™ LS Reagent (Invitrogen™) was used to extract total RNA from tissues and cells, according to the manufacturer’s protocol followed by complementary DNA synthesis using a PrimeScript RT reagent Kit with gDNA Eraser (Takara). Amplification was subsequently carried out using the GeneAmpn levels .shRNA-induced knockdown of LRH-1 expression was achieved using the lentiviral expression system. Human NR5A2 shRNA clone set was bought from GeneCopoeia (China). Viral particles were generated by co-transfecting 293T cells (ATCC) with the shRNAs and the Lenti-Pac™ Lentivirus Packaging kit , which contains packaging plasmids and a transfection reagent according to the manufacturer’s protocol. Subsequently, the shRNA viral particles transfected SKOV3 and OVCAR3 cells with 4 μg/ml polybrene (Sigma-Aldrich), and stable cell lines were established after 10 days of puromycin (2 μg/ml) selection. Knockdown was confirmed using RT-qPCR or immunoblotting. The selected cell lines were routinely cultured in puromycin-containing media until 2 days prior to experimentation.5 in 200 μl of serum-free DMEM medium) and SKOV3 cells (1 × 105 in 200 μl of serum-free DMEM medium) were seeded into the upper chamber of transwell plates in a 24-well format with 8 μm diameters . Then, 600 μl of medium containing 10% FBS was added to the bottom chamber as a chemoattractant. After 24 h of culture, cells were fixed with methanol and stained with Crystal Violet solution. The remaining cells were removed from the top of the permeable membrane using a cotton swab. Then, cells that migrated through the upper chamber were counted in four random fields under a light microscope .For migration assays, infected OVCAR3 cells (1 × 105 cells) and SKOV3 cells (1 × 106 cells) were seeded in 6-well cell culture plates and incubated for 24 h at 37°C. After achieving confluence, the cellular layer in each plate was scratched using a plastic pipette tip. The migration of the cells at the edge of the scratch was analyzed at 0 and 24 h when microscopic images of the cells were captured.OVCAR3  and supplemented with 100 μl DMEM medium with 10% FBS per well. After incubation at 37°C in a humidified incubator with 5% CO2 for 2, 24, 48, 72, and 96 hours, respectively, 10 μl of CCK-8  was added to each well. Then, after 1 h of culture, colorimetric analysis was performed on a microplate reader (Thermo Scientific™ Varioskan™ LUX) at a wavelength of 450 nm. The assay was performed using six replicates.A2780CP-sh LRH1 and A2780CP-sh NC cells were seeded in 96-well culture plates, respectively; cells were treated with CDDP (Selleck) for 48 h at 37°C at the indicated concentrations. Cell viability was determined after treatment using the CCK-8 .The chi-square test was used to analyze the statistical differences of the clinicopathologic variables. Overall survival (OS) and disease-free survival (DFS) were evaluated by the Kaplan–Meier method and log-rank test. The Cox proportional hazards model was used to estimate the independent prognostic factors for survival. Logistic regression was performed for multivariate analysis of the association between LRH1 expression and intraperitoneal metastasis. Student’s t-test was used to analyze the differences between the experimental and control groups. A two-sided P < 0.05 was considered significant.http://ualcan.path.uab.edu) is an interactive web resource based on TCGA database, which can be used to analyze relative transcriptional expression of potential genes of interest between tumor and normal samples and association of the transcriptional expression with relative clinicopathologic parameters (UALCAN (rameters . In thishttp://gepia.cancer-pku.cn/) is a web server to analyze cancer and normal gene expression and interaction (GEPIA (eraction . In thishttp://metascape.org/) is an online analytical tool, which facilitates gene annotation integration, functional enrichment, interactome analysis . However, the LRH1 mRNA expression in OC tissues with metastasis present (T2) was higher than that in T1 (P = 0.017). Moreover, LRH1 mRNA expression was more intense in the invasive foci (M) than in the in situ foci (T2) among the same patients (P = 0.034).RT-qPCR was carried out to determine the difference in the LRH1 mRNA expression between OC and normal ovarian tissues , lymph node metastasis (P = 0.011), intraperitoneal metastasis (P = 0.001), and platinum resistance (P = 0.037). LRH1 expression was not related to age, histological type, histologic grade, residual disease, ascites, and serum CA-125 level (P > 0.05).P = 0.007) and DFS (P = 0.001) compared with patients with high LRH1 (P < 0.001), histologic grade , residual disease , lymph node metastasis (both P < 0.001), and intraperitoneal metastasis  were correlated with the prognosis of patients with OC regardless of low and high LRH1 expression , residual disease (P = 0.025), and ascites (P = 0.019). Multivariate logistic regression analysis revealed that FIGO stage (P = 0.003), residual disease (P = 0.032), and LRH1 (P = 0.010) were independently linked with intraperitoneal metastasis.The traditional clinicopathological parameters of intraperitoneal metastasis were first appraised to evaluate the relationship between LRH1 overexpression and intraperitoneal metastasis (P = 0.016) and LRH1 expression (P = 0.011). Multivariate logistic regression showed that histologic grade (P = 0.026) and LRH1 expression (P \= 0.019) significantly led to lymph node metastasis  plays a crucial role in tumor invasion and metastasis in OC. We used Western blot to detect the protein levels of EMT markers, namely E-cadherin, N-cadherin, vimentin, and Moreover, we conducted CCK8 assay to evaluate whether LRH1 could mediate the effects of cell survival after CDDP treatment. Results showed that knockdown of LRH1 decreased CDDP half-maximal inhibitory concentration (IC50) in A2780CP cells  subfamily, which is expressed in the endodermal tissues of the intestine, liver, exocrine pancreas , ovary –31, pre-In vitro assays showed that LRH1 could promote tumor cell proliferation, migration, and EMT ; thus, LRH1 promoted cell migration and invasion .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.


Dietary intake influences gut microbiota activity. Nevertheless, there is a lack of evidence available that illustrates the acute effects of high glucose meal on metabolic endotoxemia. The present study assessed the acute impact of high glucose meal on endotoxemia and other clinical parameters in Saudi females with varying degrees of glycemia.2]; overweight  and T2DM . After an overnight fast, all subjects were given a standardized high-glucose (75 g) meal. Anthropometrics were taken and blood samples were withdrawn at baseline and postprandial , serum glucose, endotoxin and lipid profile were quantified.The subjects were 64 consenting pre-menopausal women, grouped into 3: control [n = 14 lean, non-T2DM, BMI = 22.2 ± 2.2 kg/mP values <.01) whereas significantly lower HDL-cholesterol levels (P < .01) were observed in T2DM subjects compared to other groups. Baseline endotoxin levels were highest in the overweight group (3.2 ± 1.1 mmol/L) as compared to control (2.0 ± 0.5 mmol/L) and T2DM (2.7 ± 1.2 mmol/L) (P = .046). HDL-cholesterol, LDL-cholesterol and triglycerides, significantly decreased in the T2DM group after 2 hours , whereas unremarkable changes observed in other groups. Lastly, endotoxin levels significantly increased only in the overweight group , 4 hours postprandial.At baseline, total cholesterol, LDL-cholesterol, triglycerides and serum glucose levels were significantly higher (High glucose meal elevates endotoxemia only among overweight subjects and impairs dysbiosis. Concurrently, several studies have examined the impact of pre-/probiotics on gut microbiota or endotoxins levels and related metabolic diseases.,18,19 Furthermore, biomedical industries have shown an intensive commercial interest in the role of the human microbiome and factors alleviating it, by using pre-/pro-biotics.,21The prevalence of obesity and diabetes across the globe has become the most devastating health impediments that have progressively increased over a decade.,23 Our previous study pointed out that metabolic endotoxemia facilitated by high-fat meal has been exacerbated in subjects with T2DM, accompanied by a parallel rise in cardio-metabolic risk profile compared to their healthy counterparts. This cardiometabolic profile includes lipids such as triglycerides, LDL- and HDL-cholesterol, levels of which are significantly linked to endotoxin-induced sub-chronic inflammation observed in patients with metabolic diseases. To the best of our knowledge, the effect of high–glucose feed on the metabolic endotoxemia, however, remains to be fully understood. Hence, the current study sought to determine the postprandial effects of a high-glucose diet on endotoxemia and other serological parameters in adult Saudi T2DM, overweight females with their healthy counterparts.It is well known that diet and feeding habits appear to play a predominant role in modulating endotoxin levels over the course of the day.22.1In this observational study, 64 Saudi pre-menopausal women were randomly recruited from different primary care centres (PCCs) in Riyadh, Saudi Arabia. A research team member was deployed to each PCC with full knowledge of the protocol to be shared with the assigned PCC physician and nurse. The study has been approved by the Ethics Committee of the College of Medicine King Saud University (No. 10–173), Riyadh, Saudi Arabia, prior to the commencement of the research and all patients gave written consent.2.2Subjects with chronic conditions such as asthma, hypertension, history of cardiac, kidney or liver disease, in addition, subjects with known long-standing diabetes and/or receiving anti-diabetic medication, those with fasting glucose levels >11 mmol/L, or with fasting triglycerides levels >4 mmol/L were excluded from the study.2.32, as well as waist-to-hip ratios (WHR) were calculated. Blood pressure (mm Hg) was measured twice using a calibrated, mercurial sphygmomanometer, with a 15-minute interval. The mean of the 2 readings was recorded. Based on BMI and DM status, consenting participants were categorized into 3 groups: Control  subjects with body mass index (BMI) 22.1 ± 2.4 kg/m2, range 7.8; overweight (n = 16) with 28.5 ± 1.5 kg/m2, range 3.6 and subjects with early onset of T2DM .Subjects were requested to visit their respective PCCSs after an overnight fast (≥10 hours). Fasting blood samples were collected at baseline to determine subjects eligibility for the study and to assess glucose control as well as lipid profiles. Weight (kg) and height (cm) were recorded using an international standard scale ). Waist circumference was measured at the level of the iliac crest at the end of normal respiration, and hip circumference was measured at the widest circumference around the buttocks using a measuring tape and body mass index (BMI) was calculated as kg/m2.4°C. All participants were given tokens of appreciation for their participation in the study.Subjects were asked to return the following day, again in a fasted state, to their respective PCC for intervention. Subjects were given a high-glucose meal similar to oral glucose tolerance test (75 g of sugar dissolved in water). They were instructed to fully consume the liquid provided immediately following baseline blood extraction. Blood samples were drawn via peripheral venous can-nula and a flushing line was added to maintain vein patency since blood was collected serially at baseline (0 hours), after 2 hours and 4 hours. Blood samples were collected and transferred to a non-heparinized tube for centrifugation. Collected serum samples were transferred to pre-labelled plain tubes, placed on ice and delivered to the chair for Biomarkers of Chronic Diseases (CBCD) laboratory in King Saud University, Riyadh, KSA, for storage at −202.5Serum glucose and lipid profile were measured routinely using an autoanalyzer . Serum endotoxin was analyzed using a commercially available QCL-1000 LAL End Point.2.6T-test was used for normally distributed variables and Wilcoxon Signed Ranks test for endotoxin. Bivariate linear regression analysis was used to determine associations between endotoxin variables of interest. Significance was set at P < .05.All statistical analyses were conducted using SPSS version 22.0 . All continuous variables were presented as mean ± standard deviation and were normalized prior to parametric analyses. For comparison between groups, the univariate general linear model (GLM) was used with Bonferroni post-hoc comparisons. Age and BMI were used as covariates. For comparison between pre- and post-intervention, paired 33.1P value <.01) and control  subjects. However, the concentration of HDL-cholesterol was significantly lower in T2DM  and overweight subjects  as compared to healthy (1.3 ± 0.3 mmol/L) subjects.Table P < .01) and control subjects . The subjects with T2DM also had significantly higher serum triglycerides (1.6 ± 0.6 mmol/L). Furthermore, endotoxin levels of the overweight subjects (3.2 ± 1.1) were significantly higher than control subjects (2.0 ± 0.5). However, the endotoxin level of T2DM subjects (2.7 ± 1.2) was not significantly different from either control or overweight subjects.As expected, subjects with T2DM had significantly higher serum glucose levels (7.3 ± 1.8 mmol/L) than the overweight+  and 2-hours levels (P < .05). Furthermore, triglycerides concentration also increased significantly at 4-hours as compared to 2-hours level (P < .05). Finally, endotoxin levels significantly increased in the overweight group at 2-hours and 4-hours as compared to baseline endotoxin after adjusting for age and use of metformin (P < .05). No significant changes in endotoxin levels were observed in both the control and T2DM group.Table 3.3P < .05)  Fig. . The res4 During this process and in the presence of a healthy gut such as the case of controls in the present study, no aberrant changes will be observed in endotoxin levels. In the case of overweight subjects, however, the significant increase in endotoxin levels following a high glucose load implicates the presence of insulin-resistance and probably systemic low-grade chronic inflammation within this cohort, making the leaky gut even leakier. Chronic exposure to endotoxin may increase T2DM risk since higher levels of endotoxin, in general, is associated with insulin resistance. However, only a few studies have evaluated the influence of nutrients and/or specific food types on endotoxemia.The main finding in the present study is that circulating levels of endotoxin are elevated after a high glucose meal among overweight non-T2DM subjects and these unfavorable changes were not observed among non-overweight, non-T2DM as well as T2DM subjects. The significant increase of endotoxin levels among overweight subjects suggests exacerbation of an already dysfunctional gut among these subjects. Physiologically, endogenous insulin released into the circulation as a response to acute hyperglycemia.,27,28 This could probably explain why endotoxin at baseline for the T2DM group is much lower than the overweight group and why the response to a glucose load was almost as similar to controls. The significant increase in endotoxin observed in the overweight group can be explained by an already existing insulin-resistant state in the subjects, as evidenced by the modest but significantly higher baseline endotoxin levels compared to other groups.In our study, endotoxin levels following a high-glucose meal showed a dramatic increase only in the overweight and not in the T2DM group. Among several possibilities, is the lack of any reduction in endotoxin levels in the T2DM group may be due to the differences in the subjects dietary make-up. The main effect, however, might be the medications that this group has been taking which include metformin. Metformin has been shown to drastically improve the gut microbiota composition among T2DM. This could explain why increasing endotoxin levels, particularly in the overweight and T2DM groups, translated to lower LDL-cholesterol levels over-all. The lack of associations between endotoxin and other cardiometabolic parameters in the present study does not supersede our previous findings on the strong relationship between endotoxin and these measures after high fat intake, and the lack of associations may be due to the sample size issues.Finally, in the present study, we observed a significant inverse association between endotoxin and post-prandial LDL-cholesterol. While no significant differences were observed between LDL-cholesterol over time in all groups, most studies indicate LDL-cholesterol levels to be lower postprandial as compared to fasting levels, at least in people with T2DM or with insulin resistance. with a larger number of subjects and inclusion of more inflammatory markers may provide clear insights into how glucose-induced endotoxemia affect the over-all metabolic picture of non-T2DM overweight subjects. Lastly, Arab women are more prone to develop obesity secondary to religious and cultural norms prevalent in the region, making them at higher risk for T2DM and therefore the study focused on this population. Whether the same altered endotoxin levels apply to men of varying glycemic levels need to be investigated separately. The study is nevertheless the first to explore the effects of high glucose load in endotoxin levels among Arab ethnic women with varying levels of glycemia. This has clinical implications, especially in the region where the concept of gut microbiota and endotoxin in particular, and how it affects obesity-related diseases, remain to be explored.The authors acknowledge some limitations and these include the small sample size and other factors not documented in the study such as lifestyle and mode of nutrition, which can significantly modify the gut microbiome. Further and longer prospective investigations using the full components of metabolic syndrome,5In conclusion, an acute high glucose load promotes endotoxemia among overweight subjects and exacerbates dysbiosis. This effect is not observed among T2DM patients on metformin as well as non-obese, non-T2DM subjects.The authors thank the volunteers and research team from the different primary care centers for the recruitment of subjects.“Conceptualization, D.A and P.T.; methodology, D.A. and M.M.E; formal analysis, S.D.H. investigation, D.A., M.G.A.A., M.M.E; S.S.; K.W.; resources, N.M.A.; data curation, K.W.; writing—original draft preparation, M.G.A.A.; writing—review and editing, S.S., P.T; supervision, N.M.A.; project administration, D.A; funding acquisition, N.M.A. All authors have read and agreed to the published version of the manuscript.Conceptualization: Dara Al-Disi, Philip McTernan.Formal analysis: Syed Danish Hussain.Funding acquisition: Nasser Al-Daghri.Investigation: Dara Al-Disi, Shaun Sabico, Kaiser Wani, Mona M. Elshafie.Methodology: Mohammed Ghouse Ahmed Ansari, Shaun Sabico, Kaiser Wani, Mona M. Elshafie.Supervision: Philip McTernan, Nasser Al-Daghri.Writing – original draft: Mohammed Ghouse Ahmed Ansari.Writing – review & editing: Dara Al-Disi, Shaun Sabico, Kaiser Wani, Syed Danish Hussain, Mona M. Elshafie, Philip McTernan, Nasser Al-Daghri.


Inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory condition of the gastrointestinal tract. Since the treatment of IBD is still an unresolved issue, we designed our study to investigate the effect of a novel therapeutic target, sigma-1 receptor (σ1R), considering its ability to activate antioxidant molecules. As a model, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to induce colitis in Wistar–Harlan male rats. To test the beneficial effects of σ1R, animals were treated intracolonically (i.c.): (1) separately with an agonist (fluvoxamine (FLV)), (2) with an antagonist of the receptor (BD1063), or (3) as a co-treatment. Our results showed that FLV significantly decreased the severity of inflammation and increased the body weight of the animals. On the contrary, simultaneous treatment of FLV with BD1063 diminished the beneficial effects of FLV. Furthermore, FLV significantly enhanced the levels of glutathione (GSH) and peroxiredoxin 1 (PRDX1) and caused a significant reduction in 3-nitrotyrosine (3-NT) levels, the effects of which were abolished by co-treatment with BD1063. Taken together, our results suggest that the activation of σ1R in TNBS-induced colitis through FLV may be a promising therapeutic strategy, and its protective effect seems to involve the antioxidant pathway system. Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing ailment of the gastrointestinal (GI) tract. Two main forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC), which are distinguished according to the area and the severity of inflammation in the GI tract. CD causes a transmural serious inflammatory damage, while UC is superficial . The theEven though the pathogenesis of IBD is complicated, several studies have demonstrated that excessive biomolecules, cytokines, chemokines, enzymes, and biochemical pathways are involved in the progression of IBD . Basical2•−), hydroxyl radicals (HO−), and reactive non-radical compounds including singlet oxygen (O2) 2•−), hyd nitrite , and -6  nitrite  are sugg2+ homeostasis [+ channel, NMDA receptors, and IP3 receptors [Sigma-1 receptor (σ1R) was discovered in 1976 by Martin et al. , who proeostasis . In respeceptors . Intereseceptors . Therefoeceptors  and cutaeceptors , while Beceptors  and NE-1eceptors .σ1R is widely studied in the brain and in neurodegenerative diseases, and is currently being suggested as a novel potential therapeutic target against inflammatory conditions . FurtherIn our present study, we presumed that the protective effect of σ1R in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis may arise from activation of antioxidant signaling pathways, primarily via GSH and the peroxiredoxin system. To the best of our knowledge, this paper is the first to study the connection between σ1R and PRDXs in IBD. Our data indicate that FLV significantly increases levels of GSH and PRDX1 antioxidants, and significantly decreases levels of 3-NT. For further confirmation, our combined treatment shows that the presence of the antagonist, BD1063, abolishes the protection exerted by FLV in the colon.As a model of colitis, 2,4,6-trinitrobenzenesulfonic acid (TNBS)  was prepared in 50% ethanol and distilled water mixture. Fluvoxamine  was dissolved in 3% dimethyl sulfoxide (DMSO). BD1063, purchased from Tocris , was dissolved in physiological saline (0.9%). The anesthetic agent thiopental  was dissolved in saline (0.9%).All experiments were performed in accordance with the standards of the European Community guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Ethics Committee  at the University of Szeged.n = 12), 50% EtOH , and TNBS . Colitis was assessed following Morris’ method [n = 6–12/group) and further treated once a day with the following drugs under mild anesthesia : fluvoxamine  i.c. administration at the previously tested effective dose 1 mg/kg dissolved in 3% DMSO); BD1063 (σ1R antagonist) 0.1 mg/kg ); FLV + BD1063 (combined administration of the two effective doses (FLV 1 mg/kg + BD1063 0.1 mg/kg)); saline (vehicle of BD1063); DMSO . The selection of the doses was done according to our previous findings in the same animal model [Male Wistar–Harlan rats (225–250 g) were purchased from Toxicoop Ltd.  and were housed in a room with an acclimatized temperature under 12 h day/night cycles with food and water ad libitum. Animals were randomly divided into three groups: absolute control , and the last 8-cm portion of the colon was removed, gently opened, rinsed in physiological saline and photographed for further macroscopic analysis. Finally, the colon segments were frozen in liquid nitrogen, powdered using a porcelain mortar and pestle, and kept at −80 °C until needed for biochemical measurements.The extent of macroscopically apparent inflammation, ulceration, and tissue disruption was analyzed in a randomized manner from the images, using a proprietary computerized planimetry software that was developed in our laboratory  and was based on planimetrics. The area of macroscopically visible mucosal damage was calculated and expressed as a percentage of the total studied 8-cm colonic segment.To determine the tissue levels of 3-NT, PRDX1, -2, -4, and -6 in the colon, we used double-antibody sandwich ELISA kits. The 3-NT kit was purchased from Bioassay Technology Laboratory , and PRDX1, -2, -4, and -6 were purchased from GenAsia Biotech Co., Ltd. . Samples were homogenized in Phosphate Buffer Saline (PBS), pH 7.4, through the same homogenization procedure ; 2 × 10 s, centrifugation = 3000 rpm, 20 min, 4 °C). The whole sample preparation procedure was done on ice. Parameters were measured according to the manufacturer’s instructions and protocols, and optical densities (ODs) were assayed at λ = 450 nm. Results are expressed in nmol/L (3-NT), pg/g protein .g, 5 min, 4 °C. Supernatants were collected and the enzyme activity in the colon samples was measured according to the manufacturer’s instructions, and ODs were determined at λ = 450 nm. From the OD values, the activity of SOD was calculated by an equation listed in the instructions and below, and expressed in inhibition %. In the equation, A = absorbance of Blank1 = 20 μL dH2O + Enzyme working solution; Blank2 = 20 μL sample; Blank3 = 20 μL dH2O; Sample well = 20 μL sample.SOD activity was measured with a kit purchased from Abcam . Samples were homogenized in ice-cold homogenization buffer . Homogenization was done using a Benchmark Scientific Handheld homogenizer D1000 ; 2 × 10 sec, centrifugation = 14,000× g for 30 min at 4 °C. Supernatants were collected and incubated in a mixture of 0.1 M CaCl2, 0.25 M sucrose, 20 mM Tris, and 1 mM DTT at 0 °C for 30 min. After incubation, centrifugation was done at 21,450× g for 60 min at 4 °C and the cytosolic fraction was used for further analyses. A solution of 125 mM Na phosphate and 6.0 mM EDTA was used as a diluent buffer for the stock solution of glutathione (GSH), glutathione reductase, 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) and β-nicotinamide adenine dinucleotide phosphate (β-NADPH). A total volume of 40 µL of each blank, standard, or colon sample, and an equal amount of DTNB stock solution (20 µL) and β-NADPH (140 µL) were added to each well, and then incubated for 5 min at 25 °C. To initiate the reaction, 10 µL of glutathione reductase was added and the absorbance was measured at 405 nm in a microplate reader after 10 min from the initiation. In the spectrophotometric assay for total GSH, GSH was sequentially oxidized by DTNB and reduced by NADPH in the presence of glutathione reductase. Total glutathione values were expressed as nmol/mg protein.In order to measure total glutathione levels in the colon, samples were homogenized in 0.25 M sucrose, 20 mM Tris, and 1 mM dithiothreitol (DTT), and centrifuged at 15,000× Protein concentration was measured by the Bradford method. Aliquots of 20 μL of the diluted samples (30× or 40× with distilled water) were taken and mixed with 980 µL distilled water. To each sample 200 μL of Bradford reagent was added, then samples were gently mixed and incubated for 10 min. Spectrophotometric measurements were taken at 595 nm and compared to a bovine serum albumin standard.p values were less than 0.05.All data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by a Holm–Sidak post hoc test  for all measurements. Differences were considered significant when the As a colitis model, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was dissolved in 50% ethanol (EtOH) and given intracolonically to rats after 12 h fasting. The dissolvent EtOH caused inflammation, ulceration, and necrotic cell death in the colon, but the colonic ulcerations due to TNBS instillation were much more pronounced. In our previous experiment, we determined the effective doses of fluvoxamine (FLV) (1 mg/kg) and BD1063 (0.1 mg/kg) in respect to testing the anti-inflammatory effect of the sigma-1 receptor (σ1R) . Here, wThe body weight change of the animals was monitored throughout our experiments. Due to colonic ulceration and therefore impaired colonic absorption, TNBS instillation was found to cause a significant body weight reduction compared to the weight change of absolute control (91.43% ± 1.77% vs. 99.46% ± 0.51%). Treatment with the σ1R agonist FLV significantly reduced the body weight loss of the animals compared to TNBS (99.40% ± 0.35% vs. 91.43% ± 1.77%). The body weights of the animals treated with FLV were similar to the absolute control group. In our combined treatment, we found that the presence of the antagonist abolished the beneficial effect of FLV according to weight loss. Furthermore, we determined that treatment with the solvents of the σ1R ligands caused no change in body weight loss compared to TNBS alone .As shown in We found that the 50% EtOH enema significantly decreased the levels of GSH compared to absolute control group (76.24 ± 6.35 vs. 106.85 ± 6.94 nmol/mg protein). TNBS significantly reduced the levels of GSH compared to EtOH (47.74 ± 2.68 vs. 76.24 ± 6.35 nmol/mg protein) compared to the absolute control group (47.74 ± 2.68 vs. 106.85 ± 6.94 nmol/mg protein). Compared to the TNBS group, the effective dose of the σ1R agonist FLV significantly elevated the levels of GSH (47.74 ± 2.68 vs. 70.19 ± 7.62 nmol/mg protein). GSH levels were decreased by the administration of BD1063, and also reduced due to the combination of FLV and BD1063 .We determined the activity of the antioxidant SOD enzyme in the colon. The induction of colitis by TNBS decreased the activity of SOD compared to the absolute control group but without significance. Treatment with FLV increased SOD activity, and BD1063 alone or in a combination with the agonist did not change SOD activity compared to the TNBS group, but our results were not statistically significant .As shown in This study suggests that σ1R activation through the administration of an agonist is protective in a chemically induced rat model of IBD, and the protective effect partially relies on the improvement of the antioxidant-oxidative stress balance. These findings are in line with the previous study of our group , which sIt has been well established that the activation of σ1R protects against ER stress and oxidative stress ,31. AdmiTNBS-induced acute colitis was developed by Morris et al. , and sinAs an oxidative stress marker, 3-NT was found in our study to increase as a result of TNBS administration. Pal et al.  found inGSH is a potent antioxidant substance that protects against oxidative stress. Pal et al.  found th2O2. In mammals, six isoforms are expressed (PRDX1-6), and PRDXs are suggested as potential targets in IBD therapy [Peroxiredoxins are highly conserved peroxidases with important roles in the elimination, sensation, and regulation of available H therapy . Accordi therapy , PRDX1 d therapy  found in therapy  suggeste therapy  found a  therapy  found th therapy  has dealBased on the current data, we suggest that the protective, antioxidant effect of σ1R, due to FLV treatment, seems to rely on the attenuation of oxidative stress. Moreover, it was shown that FLV treatment is able to activate PRDX1 isoform and GSH. This restored antioxidant balance may contribute to the anti-inflammatory and protective effect of σ1R activation against TNBS-induced colitis. Our results may offer a new therapeutic target against this serious health issue and shed light on the function and role of this enigmatic receptor.