Cholestasis is a clinical syndrome triggered by the accumulation and aggregation of bile acids by subsequent inflammatory responses. The present study investigated the protective effect of glycyrrhetinic acid (GA) on the cholestatic liver injury induced by lithocholic acid (LCA) from both anti-inflammatory and choleretic mechanistic standpoints. Male C57BL/6 mice were treated with LCA twice daily for 4 days to induce intrahepatic cholestasis. GA (50 mg/kg) and pregnenolone 16α-carbonitrile  were intraperitoneally injected 3 days before and throughout the administration of LCA, respectively. Plasma biochemical indexes were determined by assay kits, and hepatic bile acids were quantified by LC-MS/MS. Hematoxylin and eosin staining of liver sections was performed for pathological examination. Protein expression of the TLRs/NF-κB pathway and the mRNA levels of inflammatory cytokines and chemokines were examined by Western blotting and PCR, respectively. Finally, the hepatic expression of pregnane X receptor (PXR) and farnesoid X receptor (FXR) and their target genes encoding metabolic enzymes and transporters was evaluated. GA significantly reversed liver necrosis and decreased plasma ALT and ALP activity. Plasma total bile acids, total bilirubin, and hepatic bile acids were also remarkably preserved. More importantly, the recruitment of inflammatory cells to hepatic sinusoids was alleviated. Additionally, the protein expression of TLR2, TLR4, and p-NF-κBp65 and the mRNA expression of CCL2, CXCL2, IL-1β, IL-6, and TNF-α were significantly decreased. Moreover, GA significantly increased the expression of hepatic FXR and its target genes, including BSEP, MRP3, and MRP4. In conclusion, GA protects against LCA-induced cholestatic liver injury by inhibiting the TLR2/NF-κB pathway and upregulating hepatic FXR expression. Bile acids (BAs) are synthesized from cholesterol by hepatocytes and conjugated with either taurine or glycine. These primary BAs are then secreted into the duodenum from the bile duct and biotransformed by resident bacteria in the gut to secondary BAs such as lithocholic acid (LCA) and deoxycholic acid (DCA) . In the BA pool size and composition can be modulated by nuclear receptors in hepatocytes such as farnesoid X receptor (FXR) and pregnane X receptor (PXR). FXR positively regulates BSEP and OSTα/β expression and inhibits CYP7A and NTCP expression through an increase in small heterodimer partner (SHP) expression in the liver . PXR tartrigger hepatocyte apoptosis and necrosis. If left untreated, cholestasis will cause liver damage, liver fibrosis, cirrhosis, and even liver failure : a vehicle group, an LCA group, and LCA groups treated with either 50 mg/kg GA  or 45 mg/kg pregnenolone 16α-carbonitrile . The scheme of animal experiments was adapted from that of previous studies  were housed at the Department of Laboratory Animal Science, Shanghai University of Traditional Chinese Medicine in Shanghai, China. All animals were maintained on a 12-h light/dark cycle at a constant temperature (25 ± 2°C) and humidity (60%–70%). Mice were provided a standard diet and tap water  studies . The GA  studies  and our  studies . The GA  studies . Mice inThe concentrations of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), and total BA (TBA) were tested in mouse plasma using commercially available kits and a biochemical analysis system .Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm slices. The sections were subsequently stained with hematoxylin and eosin (H&E) for the evaluation of necrosis. Slides were examined under an Olympus BX41 microscope at magnifications of 40× and 400×. The necrotic score, indicating the number of necrotic foci per low-power magnification field (40×) per slide, was determined according to the method of Mouse liver tissues were extracted using ice-cold RIPA lysis buffer, and the protein concentrations were determined by a BCA protein assay kit. Equal amounts of protein extracts were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, blocked with 5% skimmed milk for 1 h at room temperature and incubated at 4°C overnight with the primary antibodies. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the proteins were detected using an enhanced chemiluminescence (ECL) method and imaged using a ChemiDoc XRS system. The densities of the bands were assessed and normalized to the β-actin or GAPDH signals. The primary antibodies were as follows: TLR2 ; TLR4 ; p-NF-κBp65 ; β-actin ; FXR ; GAPDH ; PXR ; NTCP ; OATP1A1 ; OST-β ; MRP2 ; MRP3 ; MRP4 ; and BSEP . The secondary antibodies were as follows: goat anti-rabbit  and goat anti-mouse .Gapdh was run for each sample to normalize expression levels. The relative amount of mRNA in each sample was determined using the ΔΔCT method. The sequences of the primers used are listed in Il-1β, Il-6, Tnf-α, Ccl2, and Cxcl2 mRNA levels were determined by quantitative real-time PCR (qRT-PCR). Total RNA was isolated from mouse liver tissues using TRIzol, cDNA was synthesized, and RT-qPCR was performed with primers and a 2 × SYBR Green qPCR Master Mix  in a StepOne Plus PCR System . Mouse m/z 375.1→375.1 for LCA, m/z 482.3→79.8 for TLCA, m/z 373.1→373.1 for 3keto-LCA, m/z 407.1→407.1 for CA/α-MCA/β-MCA/ω-MCA, m/z 514.3→79.7 for TCA/T-α-MCA/T-β-MCA, m/z 391.3→391.3 for CDCA/DCA/HDCA/UDCA/MDCA, m/z 498.2→79.8 for TCDCA/TDCA/TCDCA/TUDCA, and m/z 380.3→380.3 for d5-LCA. The ion spray voltage was set at −3,500 V, and the ion source temperature was set at 500°C. Nitrogen was used as the collision gas with an intensity set as a medium. Curtain gas, ion source gas 1, and ion source gas 2 were set at 25, 60, and 60 psi, respectively. Data acquisition and analysis were conducted with Analyst software .BA extraction from the liver was performed according to a previously described method . A mass p < 0.05.Data were analyzed using SPSS 21.0 software and are expressed as mean ± standard deviation. Differences between groups were calculated by one-way analysis of variance (ANOVA) and Dunnett’s test. Differences were considered statistically significant at Upon gross inspection, the gallbladder volume was increased in the LCA group compared with the vehicle group. To investigate the protective effect of GA on LCA-induced cholestatic liver injury in mice, liver slices were stained with H&E. Multiple necrotic foci and inflammatory cell infiltration to the hepatic sinus were observed in the LCA group, but the histological pattern was significantly attenuated by co-treatment with GA or PCN . The patTo compare the changes in the hepatic BAs of the mice in each group, 18 individual BAs in mouse liver were quantified by LC-MS/MS. The total amount of detected BAs  in the liver was significantly increased in the LCA group, reaching 13.33-fold higher than that in the vehicle group. In contrast, GA and PCN significantly reduced the amount of total BA, which decreased to 76.0% and 84.2% of that in the LCA group, respectively . To clarTo further compare the differences in the BA profile among each group, we evaluated the proportion of individual BAs in each group . TCA . Under normal conditions, LCA is mainly transformed into tauro-conjugates in the liver and transported into the bile duct. However, an excessive amount of LCA can cause a loss of gap junction proteins and a collapse of the bile osmotic gradient, which results in decreased movement of BAs across membranes and their subsequent accumulation in the liver . AdminisCyp3a and Sult2a to catalyze LCA hydroxylation and sulfation, which not only increase the solubility and excretion of LCA but also reduce its toxicity , 3-ketocholanoic acid (3KCA), hyodeoxycholic acid (HDCA), and CDCA by 6β-, 3β-, 6α-, and 7α-hydroxylation, respectively . Consisthe liver . Pretreahe liver . Retentihe liver . We founhe liver . MeanwhiTlr2−/− cells (data not shown). These findings suggested that the hepatoprotective effects of GA on LCA-induced cholestatic liver injury might be mediated through inhibition of the TLR/NF-κB pathway. Similar findings have been reported by In addition to reducing hepatic BA accumulation in cholestasis, the therapeutic intervention should also include the targeting of cytokine-induced inflammatory liver injury . HepatocIn summary, GA has a protective effect against LCA-induced cholestatic liver injury in mice, due to its ability to upregulate the FXR-associated pathway and prevent the activation of the TLR/NF-κB signaling pathway. PCN protects against LCA-induced liver damage by activating PXR and the downstream CYP3A11 and SULT2A1 and inhibiting the release of chemokines and cytokines by interfering with NF-κB in the liver of mice . In addi


N-acetylcysteine (NAC) were developed for providing effective intestinal mucopermeation. Polymeric ionic complexes of NAC were formed with polyethyleneimine (PEI), Eudragit E 100, and Eudragit RS 100 and loaded into a novel SEDDS. The SEDDSs exhibited a stable average size of 75 ± 12 nm (polydispersity index (PDI) < 0.3) and showed a rise in the zeta potential from −17.31 mV to −7.72 mV. On Caco-2 cells, SEDDSs at 1–3% were non-cytotoxic. An average of 91.8 ± 5.4% NAC was released from SEDDSs containing Eudragit E 100 (p ≤ 0.05) and Eudragit RS 100 (p ≤ 0.001) complexes at a significantly slower rate within 80 min, whereas the SEDDS containing PEI released NAC in a matter of seconds. Similarly, the SEDDS complexes revealed a time-dependent reduction in mucus dynamic viscosity of 52.6 ± 19.9%. Consequently, as compared with a blank SEDDS, mucodiffusion revealed about 2- and 1.8-fold significantly greater mucopermeation of SEDDSs anchoring Eudragit E 100–NAC and RS 100–NAC complexes (p ≤ 0.05), respectively. The mucoactive SEDDSs, which steadily released NAC while permeating the mucus, were linked to a significantly increased mucopermeation in vitro as a result of optimal mucolytic targeting.In this study, mucoactive self-emulsifying drug delivery systems (SEDDSs) based on sustained release of  N-acetylcysteine (NAC) affecting mucus-mucin fibers. In addition to its suitability for SEDDS incorporation in this study, NAC as a small molecular weight thiolated molecule is a potential prototype mucolytic agent [Self-emulsifying drug delivery systems (SEDDSs) are a platform oral dosage form, which allows for the solubilization of a wide range of pharmaceuticals. When these systems come in contact with gastrointestinal (GI) fluids, on the one hand, they spontaneously create an oil-in-water emulsion, providing a suitable method of drug administration delivered via the intestinal mucosal route ,2. Intesic agent ,11. Howeic agent ,13, repric agent ,15. Accoic agent ,14. Adeqic agent ,17,18,192−), N-acetylcysteine (NAC), macrogolglycerol ricinoleate (Kolliphor EL), fluorescein diacetate (FDA), sorbitan trioleate (Span 85), and dipropylene glycol were purchased from Sigma-Aldrich . Evonik Röhm GmbH  gifted the methacrylate-copolymers Eudragit® E 100 (poly(butyl methacrylate-co-(2-demethylaminoethyl) methacrylate-co-methyl methacrylate), 1:2:1) and Eudragit® RS 100 . 2-n-Octyl-1-dodecanol as an oil phase was purchased from Tokyo Chemical Industry Co., Ltd. . The remaining materials and solvents were of analytical grade and purchased from commercial sources.Branched-polyethyleneimine , polyoxyethylene (20) sorbitan monooleate (Tween 80), 5,5′-dithiobis-(2-nitrobenzoic acid) . Ellman’s buffer at 0.5 M and pH 8 comprising 0.05% DTNB2− was used to dilute the supernatants and reference solutions omitting polymers. The dilutions were both transferred to a UV microplate reader to measure the absorbance determined at 450 nm. Absorbances indicating the presence of NAC were utilized in Equation (1) and the precipitation percentage was calculated. Acetone wash was followed for NAC precipitates represented by PEI and Eudragit E 100 complexes that were subsequently brought to immediate dryness. Moreover, precipitates of the Eudragit RS 100–NAC complex were lyophilized at −80 °C following demineralized water wash. Mucoactive SEDDSs were developed based on incorporating these complexes:NAC hydrophobic ionic complexes with different polymers, listed in Ionic complexes were characterized using scanning electron microscopic (SEM) imaging. Following the dissolution of complexes in methanol, 10 µL droplets of the solubilized complexes were placed on carbon tapes and incubated at 25 °C until dryness. Complex droplets adsorbed to carbon tape using Polaron SEM coating unit E5100 were coated with a 10 nm thick gold layer. Samples were placed in a JEOL JSM-6010 LV scanning electron microscope for imaging. The SEM images of the acquired NAC polymer ionic complexes are shown in −1 wavenumber and a speed of 4 cm−1, solids were placed on the tip of a platinum attenuated total reflection instrument and scanned 32 times. Bruker ALPHA Fourier transform infrared spectroscopy  was used to record IR spectra for NAC, polymers, and their complexes. Using a range of 4000–400 cmv/v) each, as adapted from previous works reporting SEDDSs [2− at pH 6.8 in 10 mL final volumes. Dilutions were placed on an Eppendorf Thermomixer  at 37 °C and 300 rpm shaking force. Afterward, 1 mL aliquots were taken at 0 h and 4 h and transferred to a particle sizer  to measure the SEDDS droplet’s size and polydispersity index (PDI). SEDDS droplet’s zeta potential was measured after dilution in 10 mM phosphate buffer medium with pH 6.8. The novel mucoactive SEDDSs in this study were developed by mixing various excipients with the obtained complexes described above. Firstly, the surfactants Tween 80, Kolliphor EL, and Span 85, the cosolvent dipropylene glycol, and the oil phase 2-n-octyl-1-dodecanol were blended at 20%  under optimal incubation conditions at 37 °C and 5% CO2. MEM replenishment was considered regularly until thick Caco-2 cell monolayers were reproduced. In 25 mM HEPES-buffered saline (HBS) at pH 7.4, SEDDSs containing the NAC polymer ionic complexes were diluted at 1:100 and 3:100. Positive and negative controls were HBS as a blank and 0.05% (mass/vol.) Triton-X® 100 solutions, respectively. Under a light microscope, Caco-2 cell layers were confirmed, and thereafter, cells were washed thrice with preheated (37 °C) HBS. Caco-2 cells were incubated for 4 h with the positive and negative controls, as well as the tested SEDDS dilutions were added in triplicate at 500 µL/well. After that, each well was washed twice with prewarmed HBS, and 250 µL of 2.2 µM resazurin solution was added to each well. Following a 3 h incubation period at 37 °C and 5% CO2, aliquots of 100 µL were transferred to 96-well black microplates, and fluorescence was quantified with a microplate reader utilizing excitation (540 nm)/emission (590 nm). According to Equation (2), cell viability was defined as a function of fluorescence recovery based on viable cells’ abilities to produce the pinkish resorufin by reducing resazurin as compared with 100% fluorescence produced by the positive control:Using the resazurin test, biocompatible SEDDS concentrations on the Caco-2 cell line were determined. Briefly, Caco-2 cells at 2.5 × 102−-containing 0.1 M sodium phosphate buffer solution at pH 6.8. Increasing amounts of the NAC polymeric ionic complexes were completely dissolved in all phases. Free NAC dissolution was used as a control. All samples were kept for 24 h under 550 rpm shaking force at 37 °C using a thermomixer. All phases after producing clear solutions were separately aliquoted in Eppendorf tubes (2 mL) among which the n-octanol phase was lyophilized. Aliquots from all phases were diluted in sodium phosphate buffer (0.5 M at pH 8) containing 0.05% DTNB2− at the same final volume considering predissolution of the n-octanol phase leftover in acetone that was subsequently evaporated. Following 4 h of additional shaking at 37 °C, the diluted samples were centrifuged, sub-aliquoted (100 µL), and their sub-aliquots were analyzed regarding absorbance at 450 nm via a microplate reader. The obtained absorbances of the dissolved NAC were substituted in Equations (3) and (4) to calculate the partition coefficients.The solubility test was performed in two phases: the lipophilic phase represented by n-octanol and SEDDS excipients, and the aqueous phase represented by deionized water and 0.05% DTNB2− or Ellman’s reagent was dissolved at a concentration of 0.05% in 0.1 M sodium phosphate Ellman’s buffer solution at pH 6.8 was employed as a thiol detector upon NAC release from SEDDSs. Ellman’s reagent ) undergoes a disulfide–exchange reaction with thiol substructures to form 2-nitro-5-thiobenzoate dianion (TNB2−), a spectrophotometrically sensitive product that quantitatively measures how much sulfhydryl groups reacted in a solution [2− in Ellman’s buffer prevents its reverse diffusion into SEDDS droplets, and thus, permits the disulfide exchange with the released NAC only. As a result, this quantification method provides a real-time selective disulfide exchange process, signifying its appropriateness for NAC detection. ®, Tecan Trading AG, Zurich, Switzerland) was used to measure the release of NAC from the SEDDSs at each time point at a wavelength of 450 nm. The reference positive control utilized methanolic NAC polymer ionic complex solutions diluted in the release medium. The uprise in the absorbance from SEDDS dilutions detected at each time-point was substituted in Equation (5) to figure out the percentage of the released NAC from SEDDSs in correlation to the reference 100% control. To predict NAC release, the Nernstsches equation (Equation (6)) [SEDDS/release medium found in Equaiton (3) as follows:The thiol substructure of NAC was utilized in Ellman’s assay to feature its release from the SEDDSs . For thisolution . Furtherion (6))  was usedNAC was dissolved using different dilutions in the release medium at concentrations ranging from 0.0025 to 0.02%, to address the above released NAC from SEDDSs. A spectroscopic analysis to read absorption at 450 nm was performed on all dilutions that were aliquoted in triplicate using a microplate reader.The rheological changes on intestinal porcine mucus influenced by the released mucolytic NAC from SEDDSs were in vitro analyzed via a rheometer . Following a previous protocol, mucus dynamic viscosity (η), elastic modulus (G′), and viscous modulus (G″) were investigated . Collect2 and pore size of 3 µm  were used and these inserts were covered with 50 mg of intestinal mucus. The upper donor compartment of inserts attaching a tightly sealed filtering membrane (pore size = 3 µm) at the bottom was gently filled with 250 µL of pH 6.8 phosphate buffer (0.1 M) containing 1:100 dilutions of the tested SEDDSs. Conversely, the acceptor compartment directly underneath the filtering membrane held 500 µL of the same blank buffer. Wells comprising the positive and negative controls had donor compartments filled with a blank SEDDS and an FDA-labeled SEDDS diluted in buffer, respectively, in absence of the mucus. The test and control samples were incubated for 4 h on a plate shaker with a gentle 75 rpm shaking force at 37 °C and experimented in triplicate. During this, 100 µL aliquots on an hourly basis were drawn from all of the acceptor compartments with fresh buffer replacement. Aliquots were transferred to 96-well plates and mixed with 10 µL of 5 M NaOH to ensure FDA hydrolysis and fluorescein release following additional incubation for 30 min. The cumulative fluorescence detected instrumentally at 480 nm/520 nm (excitation/emission) using a microplate reader was used to calculate the percentage of the permeated SEDDSs.The static transwell mucodiffusion model was used in vitro to evaluate mucoactive SEDDS diffusion through the intestinal mucus. Quantification of SEDDSs permeating the mucus was aided with a fluorescence tracer labeling the permeated SEDDSs. For this aim, the mucoactive and blank SEDDSs were labeled with the model drug FDA using acetone. An acetone solution of FDA at a concentration of 10 mg/mL was formed and pipetted at a volume of 100 µL into each tube containing 500 µL of SEDDSs. In reference to The permeated mucoactive SEDDS droplets from the transwell model above were confirmed using SEM images following the method mentioned earlier . AliquotThe ability of SEDDSs to remain resident on intestinal mucosa was measured quantitatively in vitro over a specific time using a previously developed method . Plasticp-value ≤ 0.05). Significant differences between data points were classified as * p ≤ 0.05 (significant), ** p ≤ 0.01 (very significant), and *** p ≤ 0.001 (highly significant). The linear regression square (r2) describing the line graph of different NAC concentrations in GraphPad Prism V. 7.05 was used for statistical analysis. Two-way ANOVA described experimental significances using the 95% confidence interval  and ≤40 min (p ≤ 0.05), respectively. In addition to the aforementioned sufficient lipophilic property of Eudragit E 100 and RS 100, these two polymers as per molecular unit possess a large molecular weight with around 188 and 10 amino cationic binding sites, respectively. Thus, they successfully trapped NAC in the SEDDSs through ionic bonding that significantly enhanced its lipophilicity and decreased its release rate. Eudragit RS 100’s permanent cationic ammonium substructures, however, generated stronger ionic bonds and provided more sustained NAC release than Eudragit E 100’s pH-dependent tertiary amine [The partition coefficient (log D) characterizing in situ drug release is primarily the most reliable model describing in vitro release kinetics from SEDDSs . Howeverry amine . Nazir ery amine .2−-preserving activity induced an instantaneous disulfide–exchange reaction at pH > 6, allowing for the monitoring of time-dependent NAC release [2− that formed a disulfide heterodimer with NAC subsequent to its entire release from SEDDS complexes. This was obtained by comparing the molar equivalence of DTNB2− required to react with the quantified thiol substructure from NAC upon its presence in release medium. NAC that released nearly in full amount, highlighted about ~344.6, 232.2, and 118.8 µg/mL of reacted DTNB2− with respect to NAC that released from PEI’s, Eudragit E 100’s, and Eudragit RS 100’s SEDDS complexes, respectively. Only by releasing NAC with a sulfhydryl group having a pKa of 9.8 [2− be formed [2− and caused considerable yellow staining in the release medium. Provided that optimum NAC reactivity was attained from the release studies, the likewise disulfide–exchange interactions between the released NAC and mucus-cysteine subdomains could also predict significance.Designing SEDDSs that regularly emit numerous low mucolytic bursts of a loaded mucolytic structure is a successful technique for SEDDS optimal mucodiffusion . In addi release ,44. Figua of 9.8  can a nee formed . Based o2− disulfide reaction  than the control blank SEDDS. Therefore, mucoactive SEDDS reactivity to mucus to improve overall mucopermeation in this study correlated better with SEDDSs that slowly release their mucolytic load. In turn, this could explain why mucopermeation did not improve much for the PEI–NAC complex-loaded SEDDSs that exhibited immediate release of NAC. Due to the lack of sufficient lipophilic enhancement of free NAC in SEDDS droplets, a previously reported NAC load of 3% in SEDDSs showed minimal or no improvement in mucodiffusion. However, the same study reported equimolar novel thiol conjugates in SEDDSs namely, thiobutylamidine-dodecylamine (log P = 5.83) and thioglycolic acid-octylamine (log P = 3.14), with significantly improved mucodiffusion [p ≤ 0.001) greater levels of mucoactive SEDDS residing in the intestinal mucosa after a 4 h incubation period, highlighting the mucolytic load of mucoactive SEDDSs containing Eudragit E 100–NAC, and Eudragit RS 100–NAC complexes, respectively. Unlike the lipophilic Eudragit E 100–NAC and RS 100–NAC complexes, the PEI–NAC complex’s SEDDSs as comparedwith the blank SEDDS exhibited a 1.3-fold higher amount of SEDDS droplets left on the intestinal mucosa that was non-significant (p > 0.05). This is because the immediately released NAC out of this SEDDS complex has non-significant mucolytic action and is washed away by the buffer medium’s continual flow. According to Leichner et al., using the mucolytic activity of lipidized papain enzyme ionically coupled with sodium deoxycholate and anchored in SEDDSs, the fraction of SEDDSs that remained resident on porcine intestinal mucosa was increased by twice as compared with unloaded SEDDSs. Using the transwell diffusion technique, they also demonstrated that increased mucosal residence of mucoactive SEDDSs was associated with a two-fold increase in SEDDS mucopermeation [SEDDS mucopermeation was addressed via the static in vitro transwell model by quantifying the amount of SEDDSs that diffused between two compartments, the donor and acceptor compartments separated by a mucus layer . Figure iffusion . As a reiffusion , and henrmeation . In thisp ≤ 0.001) from Eudragit RS 100 and Eudragit E 100 (log D > 5) as compared with PEI or free NAC (log D ≤ 0.5) that exhibited an immediate NAC release. Parallel to the NAC release behavior, intestinal mucus viscoelastic parameters influenced by the SEDDSs loading the NAC ionic complexes as compared with the blank SEDDS revealed considerably lower dynamic viscosity, elastic modulus, and viscous modulus. Finally, as compared with PEI–NAC complex-loaded SEDDS, mucoactive SEDDSs anchoring Eudragit E 100–NAC and RS 100–NAC complexes displayed significantly higher amounts of SEDDSs that permeated the mucus (p ≤ 0.05) and significantly improved SEDDS intestinal mucosal residence (p ≤ 0.001). Based on these findings, a significantly improved in vitro mucopermeation was achieved by mucoactive SEDDSs that showed adequate NAC sustained release. The resultant droplets of these SEDDSs were shown to produce better targeting mucolytic effect, meanwhile traversing mucus layers.The size (<100 nm), PDI (<0.3), and zeta potential (average = −7.72 mV) stability among the produced mucoactive SEDDSs containing NAC polymer ionic complexes were all preserved in this study. The resazurin assay, which showed >85% cell viability, validated biocompatible and non-toxic SEDDS concentrations when applied on Caco-2 cells at 1% and 3% dilutions. SEDDSs demonstrated that NAC leaking out of droplets released at a significantly slower rate (


Ih) in dorsal hippocampal CA1 neurons, the underlying molecular mechanisms are entirely unknown. Following chronic social defeat stress (CSDS), susceptible mice displayed social avoidance and impaired spatial working memory, which were linked to decreased neuronal excitability, increased perisomatic hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 protein expression, and elevated Ih in dorsal but not ventral CA1 neurons. In control mice, bath application of corticosterone reduced neuronal excitability, increased tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b) and HCN1 protein expression, and elevated Ih in dorsal but not ventral CA1 region/neurons. Corticosterone-induced upregulation of functional Ih was mediated by the glucocorticoid receptor (GR), HCN channels, and the protein kinase A (PKA) but not the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Three months after the end of CSDS, susceptible mice displayed persistent social avoidance when exposed to a novel aggressor. The sustained behavioral deficit was associated with lower neuronal excitability and higher functional Ih in dorsal CA1 neurons, both of which were unaffected by corticosterone treatment. Our findings show that corticosterone treatment mimics the pathophysiological effects of dorsal CA1 neurons/region found in susceptible mice. The aberrant expression of HCN1 protein along the somatodendritic axis of the dorsal hippocampal CA1 region might be the molecular mechanism driving susceptibility to social avoidance.While chronic stress increases hyperpolarization-activated current ( Post-traumatic stress disorder (PTSD) is a debilitating long-term mental disease that can develop following exposure to traumatic, life-threatening events such as physical, emotional, or sexual attacks, natural disasters, wars, and pandemics . RelivinAnimal models are critical for researching the pathophysiology of PTSD because they allow researchers to track the development of PTSD over time using controlled stresses. It is worth noting that it only affects a small percentage of persons who have been exposed to potentially fatal events. Chronic social defeat stress (CSDS) is a psychosocial stress animal model that causes PTSD symptoms like social avoidance, emotional shifts, and anxiety-like behavior . CSDS prFunctional neuroimaging of PTSD patients reveals structural and functional changes in the hippocampus, amygdala, and medial prefrontal cortex –17. In P+ and K+ permeability, and (3) activation by membrane hyperpolarization [Ih in the dorsal but not ventral CA1 region/neurons [Ih, on the other hand, are unknown.HCN channels generate membrane currents that are characterized by (1) cyclic nucleotide-mediated modulation, (2) Narization . HCN1 isrization , 37. In rization , 39. A prization . Evidencrization –46. Wholrization . Normal rization . Rats ex/neurons , 44. Thein) and reduced neuronal excitability. In susceptible mice, perisomatic HCN1 protein expression and Ih were elevated in the dorsal but not ventral CA1 neurons/region, indicating increased functional Ih . In control mice, bath application of corticosterone reduced Rin and decreased neuronal excitability in the dorsal but not ventral CA1 neurons. We further found that corticosterone pretreatment increased Ih as well as TRIP8b and HCN1 protein expression in the perisomatic CA1 region/neurons of the dorsal hippocampus. Corticosterone-induced functional Ih was mediated by the GR, HCN channels, and the PKA but not the CaMKII pathway. Susceptible mice were reintroduced to novel aggressor mice one month or three months after the end of the CSDS and displayed persistent social avoidance. This long-term behavioral impairment was linked to decreased neuronal excitability, increased TRIP8b and HCN1 protein expression, and elevated Ih in the dorsal CA1 region/neurons. On the other hand, resilient mice displayed increased neuronal excitability in the dorsal CA1 neurons. Corticosterone had no additional impacts on functional Ih in the dorsal CA1 neurons from susceptible mice, but it did in control and resilient mice. Our findings show that functional Ih may be involved in cellular pathways that drive the development of susceptibility and resilience to social avoidance.This study found that CSDS produced susceptible and resilient phenotypes during the social interaction test. Although both susceptible and resilient mice showed anxiogenic-like behavior during the elevated plus-maze test, spatial working memory was impaired in susceptible mice compared to control and resilient mice in the Y maze spontaneous alternation test. Compared to control and resilient mice, dorsal but not ventral CA1 neurons had lowered input resistance  were used in this study. Mice were housed 5 per cage on a 12 h light schedule (on 7:00 A.M. off 7:00 P.M.) with ad libitum access to water and food. In a study with one month or three months without CSDS, mice were single-housed following CSDS. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Augusta University.Corticosterone (Cat # 3685), dexamethasone(Cat # 1126), mifepristone , KT5720 (Cat #1288), KN-62 (Cat #1277), and ZD7288 (Cat #1000) were purchased from Tocris. Corticosterone, Dexamethasone, KT5720, and KN-62 were dissolved in dimethyl sulfoxide and diluted to a final concentration in artificial cerebral spinal fluid (aCSF).Social defeat was generated using a resident–intruder paradigm with minor modifications . 3-to-4-All behavioral measurements were analyzed by custom-written programs. All behavioral experiments were performed between 4 pm and 6 pm. All experiments were performed under blind conditions.Social interaction test was assessed at 48 h after the last social defeat stress session. Mice were acclimated to a behavior room under red-light conditions (~4 lux) for at least 1 h. The apparatus is made of a box (18 × 18 × 12 in) with a perforated Plexiglass cage (4 × 4 × 12 in) at the middle of one side of the box. The social interaction test consisted of a two-trial procedure under red-light conditions (~4 lux). The experimental mice were placed in the corner of the open arena and allowed to freely explore for 2.5 min . After the 1st session, an unfamiliar CD1 male mouse was introduced into the perforated Plexiglass cage as a social stimulus for 2.5 additional min . The surface of the open field was cleaned with 70% EtOH in order to remove permeated odors by previous animals after each trial. A CCD camera placed above the apparatus recorded behavior. A social interaction ratio was calculated as the ratio of the time spent in the interaction zone in the presence of a social target to the time spent in the interaction zone without a social target. Mice with scores more than 1 were defined as “resilient” and those with scores less than 1 were defined as “susceptible” .Anxiety was tested on the elevated plus-maze apparatus described previously . The appo angle from each other. In the Y maze, there is no blocking off of each arm. The mice were placed in the one arm of the Y maze (start arm) and allowed to freely explore the arena for 6 min. The sequence of arm entries and the total number of arm entries were measured. The surface of the maze was cleaned with 70% EtOH in order to remove permeated odors by previous animals after each trial. A CCD camera placed above the apparatus recorded behavior. Arm entry is defined as having all four paws in the arm. An alternation is defined as successive entry into three different arms, on overlapping triplet sets. The percentage of alternations was calculated as the number of actual alternations divided by the maximum number of alternations .Short-term spatial working memory was examined using the Y maze spontaneous alternation test as previously described . The app2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 7 dextrose, 210 sucrose, 1.3 ascorbic acid, and 3 sodium pyruvate, bubbled with 95% O2 − 5% CO2. The brain was removed and hemisected along the longitudinal fissure. Dorsal  and ventral  hippocampal slices were prepared as previously described [2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, 12.5 dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate, bubbled with 95% O2 − 5% CO2 at 35 °C for 30 min and then incubated for at least 45 min at room temperature before used for electrophysiology.Mice were anesthetized with a lethal dose of isoflurane (>5%) in a closed plastic container and transcardially perfused with ice-cold aCSF composed of (in mM): 2.5 KCl, 1.25 NaHescribed . Hippoca2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, and 12.5 dextrose, bubbled with 95% O2 − 5% CO2 at a rate of 1 ml/min and 31–33 °C. For whole-cell voltage-clamp recordings, dorsal hippocampal slices were transferred to the recording chamber and perfused with aCSF containing (in mM) 125 NaCl, 3 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, and 12.5 dextrose, 1 BaCl2, 10 TEA-Cl, 0.2 3,4-DAP bubbled with 95% O2 − 5% CO2 at a rate of 1 ml/min and 31–33 °C. CA1 pyramidal neurons were visually identified using a microscope  fitted with differential interference contrast optics [Whole-cell current-clamp recordings were performed as previously described , 42, 43.t optics . Patch p2, 1.0 MgCl2, 20 TEA-Cl, 0.2 3,4-DAP, 1 BaCl2, pH 7.3 with KOH. Membrane currents were recorded using a MultiClamp 700B amplifier  and commercial acquisition software pCLAMP10 . Electrical signals were sampled at 10 kHz, analog filtered at 2 kHz by using Axon Digidata 1440 A (Axon Instruments). Maximal Ih was measured by using hyperpolarizing voltage commands to −140 mV from a holding potential of −30 mV. Linear leakage and capacitive currents were digitally subtracted by scaling traces at smaller command voltages in which no voltage-dependent current was activated.Cell-attached voltage-clamp recordings were performed as previously described , 44. PipNeuronal reconstructions were performed as previously described . BrieflyImmunohistochemistry was carried out as described previously , 43. 300Western blotting was carried out as previously described , 43. Aftdv/dt (mV/msec). AP threshold was defined as the voltage where dv/dt of the spike crossed 20 mV/ms. AP amplitude was determined from threshold to peak, with the half-width measured at half this distance.Input resistance was measured by the slope of the linear fit of the V-I plot between +30 and −150 pA current injections. Single action potentials (APs) were analyzed for AP threshold, AP amplitude, AP half-width, and maximum t-test (Wilcoxon signed-rank test) or unpaired t-test (Mann-Whitney U test) with GraphPad software. The results of all statistical analyses were compiled into an word file. *P < 0.05 was considered as statistically significant.Statistical comparisons were performed using ANOVA (one-factor or two-factor) followed by Bonferroni post-hoc test, paired We used male mice aged 6 to 7 weeks and subjected them to CSDS for 12 days. Figs.  and S1A.in of dorsal CA1 neurons was considerably lower in susceptible mice compared to the control and resilient mice at resting membrane potential (RMP)  Fig.  and at −MP) Fig. , B. The MP) Fig. , implyinMP) Fig. . RMP (FiMP) Fig. , Rin at RMP Fig. , Rin at  mV Fig. , D, and  mV Fig. , on the 1 region , 55, CA1in [Ih at the soma of dorsal CA1 neurons using cell-attached patch-clamp recordings. After subtracting linear leakage and capacitive currents by scaling traces at −40 mV, the h current exhibited a lack of time-dependent component of Ih in response to voltage, as shown in Fig. Ih than patches from the control and resilient mice  of the h channel activation curve for susceptible group was significantly shifted to the right by around +10 mV  pumps in CA1 neurons increases perisomatic Ih [in and the number of action potentials) after 20 min, and the effects were reversible over time (Data not shown). We, therefore, treated corticosterone for 20 min in all our experiments. In dorsal CA1 neurons, corticosterone had no effect on RMP  with different concentrations of corticosterone . We found that neither 10 nM corticosterone  in dorsal CA1 neurons was due to the increased HCN channel trafficking. We used a 50 μm thick resection from either vehicle- or corticosterone-pretreated dorsal hippocampal slices (300 μm thick) for immunohistochemistry  [in at RMP  is a potent, selective inhibitor of PKA signaling [Ih. Rin at RMP  , 68. We  4.6 nM) . We testRMP Fig. , C, and RMP Fig. , E were MP Figs. , Rin at  mV Fig. , and act mV Fig. , J witho mV Fig. , G. RU 4RMP Fig. , and at RMP Fig. . When doMP Figs. , and at MP Figs. . FurtherMP Figs. , E. Thes effects , 72. In rylation . We, theignaling . As prevignaling , 75. In MP Figs. , Rin at  mV Fig. , and the mV Fig. , E were tivation , we nextf 0.9 μM . 5 μM KNf 0.9 μM , 77. We,RMP Fig. , Rin at  mV Fig. , and the mV Fig. , E folloto 20 μM , 44. The20 μM [m . To circRMP Fig.  and at −RMP Fig.  comparedRMP Fig. , E, Rin MP Figs. , Rin at  mV Fig. , and the mV Fig. , H.Ih in dorsal but not ventral CA1 region/neurons. We, first, examined if susceptible mice exposed to novel aggressor mice re-experienced the trauma during the social interaction test. Because the difference in human age between mice aged 3 months and 6 months is over 10 years [Ih. CSDS was subjected to male mice aged 6 to 7 weeks for 12 days  were altered between groups. RMP was unaffected by corticosterone in any of the groups  in hippocampal and cortical pyramidal neurons [in and neuronal excitability. However dorsal CA1 neurons from resilient mice had higher Rin and neuronal excitability than control and susceptible groups  [2+ current amplitude in rat/mouse hippocampal CA1 neurons [in and lower neuronal excitability following corticosterone treatment   following corticosterone treatment, whereas control and resilient mice did. These findings imply that elevated Ih in the dorsal CA1 neurons from susceptible mice occluded corticosterone-induced upregulation of functional Ih.We initially examined the neuronal excitability of dorsal and ventral CA1 neurons in control, susceptible, and resilient male mice since hippocampal hypoactivity has been linked to human PTSD . After 1sistance . This pa neurons . Surprisups Fig. . Compare amnesia –87. Norm amnesia . It has  amnesia , 89. Conability) , 63. Cor neurons , 62. Indent Fig. . Dexametked Fig. . Further88) Fig. . These fendrites , 39. Theendrites , 44, 90.ductance . When doked Fig. . As a reIh and a decrease in neuronal excitability) [Selective serotonin reuptake inhibitors are Food and Drug Administration-approved medications for treating PTSD that primarily act by targeting the monoamine system and raising serotonin levels in limbic brain regions, such as the hippocampus. However, after the initial pharmacologic treatment, only around 20–30% of patients with PTSD satisfy remission criteria , 92. Desability) , 44. A rability) . Corticoability) , 44.Ih in dorsal CA1 neurons from susceptible mice, while it affected the control and resilient groups. Our findings imply that abnormal subcellular expression of HCN1 protein along the somatodendritic axis of dorsal CA1 neurons contributes to a reduction in neuronal excitability and may be the molecular mechanism underpinning the development of susceptibility to social avoidance.In conclusion, we have demonstrated that susceptible mice displayed social avoidance and impaired spatial working memory. In dorsal CA1 neurons, susceptible mice reduced neuronal excitability, whereas resilient mice had increased neuronal excitability. These results suggest that the neuronal excitability of dorsal CA1 neurons might be the cellular mechanism underlying the development of susceptibility and resilience to social avoidance. Acute corticosterone treatment in dorsal CA1 neurons from control mice, where perisomatic GR was strongly expressed, produced comparable effects, as seen by susceptible mice. These effects of corticosterone were mediated by the GR, HCN channels, and the PKA pathway. Importantly, corticosterone did not affect functional Supplementary FiguresSupplementary Table 1Statistical Table


Under these conditions, the ellagic acid yield was 6.30 ± 0.92 mg/g pomace with an antioxidant activity of 79.0 ± 0.96 μmol Trolox eq/g pomace (DPPH assay), which are 2.37 and 1.32 times the values obtained by extraction with methanol–acetone–water solvent, respectively. The considerable improvement in ellagic acid extraction efficiency could be highly attributed to the reactions of lipid saponification and ellagitannin hydrolysis resulted from sodium bicarbonates. The present study has established an organic solvent-free method for the extraction of ellagic acid from raspberry wine pomace, which is feasible and practical in nutraceutical applications.Industrial processing of raspberry juice and wine generates considerable byproducts of raspberry pomace. Ellagic acids/ellagitannins, being characterized by their antioxidant and antiproliferation properties, constitute the majority of polyphenolics in the pomace and are valuable for recovery. In the present study, we developed a novel procedure with sodium bicarbonate assisted extraction (SBAE) to recover ellagic acid from raspberry wine pomace. Key parameters in the procedure, i.e., sodium bicarbonate concentration, temperature, time and solid/liquid (S/L) ratio, were investigated by single factor analysis and optimized subsequently by Response Surface Methodology (RSM). Optimal parameters for the SBAE method here were found to be 1.2% ( Rubus idaeus L.) fruit is a well-known red juicy berry, belonging to the Rosaceae family and distinguishing itself by high ellagitannin contents compared to other plants in the same family, e.g., blackberry and strawberry [https://www.fao.org/faostat/en/#data/QCL/visualize, accessed on 2 February 2022), and industrial processing of raspberries into juice or wine generates 10–12% (w/w) pomace as byproduct [The raspberry  to determine the compound concentrations. The solvent used for extraction could be generally allocated into three categories: neutral organic ,4,34, acIn this study, a novel method to extract ellagic acid from raspberry wine pomace with sodium bicarbonate was developed. The key processing parameters, i.e., sodium bicarbonate concentration, temperature, extraction time and solid/liquid (S/L) ratio, were firstly investigated with single factor analysis, followed by subsequent optimization by Response Surface Methodology (RSM). The optimized conditions were further validated by examining the ellagic acid extraction rate and its antioxidant activity. Finally, potential mechanisms of sodium bicarbonate assisted extraction (SBAE) were discussed.In our study, 2.66 ± 0.35 mg/g ellagic acid was extracted from the milled raspberry pomace by methanol–acetone–water (MAW) method, while 1.98 ± 0.12 mg/g ellagic acid was obtained by acetone–acetic acid–water (AAW) method. This difference could be partially explained by the S/L ratio difference, which was in total 1:45 for MAW and 1:20 for AAW.p < 0.001), the extraction method assisted with analytical sodium bicarbonate was further studied.Except for the baking soda, the ellagic acid concentration extracted by the other food additive solutions ranged from 3.43–16.7 mg/L, and the yield varied between 0.024 and 0.117 mg/g, while the baking soda reagent achieved 45.8 ± 0.8 mg/L concentration and 0.321 ± 0.006 mg/g yield . As ella3 concentration, temperature, time and solid/liquid (S/L) ratio  ratio . When exectively A. Temper.28 mg/g B. As for mg/g  and a time of 20 min. Regression and ANOVA analysis demonstrated that the relationship between the ellagic acid yield and extraction parameters is quadratic with good regression coefficient (R2 = 0.9293). The model significance was confirmed by an F value of 10.22 and a p value of 0.003 , but X1, X3, X1X2, X1X3, X2X3 were not (p > 0.05), suggesting NaHCO3 concentration plays a more important role than the other two parameters for extraction efficiency. Judging from the quadratic polynomial model, the optimal parameters for SBAE were 1.2% NaHCO3, an S/L ratio of 1:93 and an extraction time of 22 min with the predicted ellagic acid yield to be 6.277 mg/g. Extraction experiments were performed in triplicates using the parameters above, and the resulting ellagic acid yield was determined as 6.295 ± 0.917 mg/g, revealing that there was no significant difference compared to the prediction.The response surface plots about the effects of NaHCOof 0.003 D, and thp < 0.001), which was consistent with the yield difference. However, antioxidant activity derived from the FRAP test showed the MAW extracts to be the highest, with the AAW and SBAE being similar (p = 0.003). One of the possible reasons for the low FRAP activity in SBAE extracts could be that the FRAP test required an acidic environment, which may interact with the NaHCO3 reagent contained in the SBAE extracts, causing a relatively low result. However, a DPPH activity of 79.0 ± 0.96 μmol Trolox eq/g and a FRAP activity of 0.631 ± 0.075 mmol Fe(II)S eq/g were still comparable to those in other studies that conducted organic solvent extraction from raspberry seeds [The antioxidant activities of SBAE extracts under optimal conditions, as well as those obtained by MAW and AAW, were measured by DPPH  and FRAP (ferric reducing antioxidant power) methods . DPPH scry seeds , which sThe raspberry wine pomace in this study was a dark pink color and was rich in raspberry seeds A. The SBSeveral studies have conducted ellagic acid or ellagitannin extraction with organic solvent from the whole fruit or seeds. For instance, Agnieszka et al.  achievedAlthough food additive solutions have been rarely used in polyphenolic extraction, they were tested in the present study due to the following two reasons: (1) to minimize the application of organic solvents, and (2) to improve the solubility of ellagic acid in the extraction solution. Generally, maltodextrin, xylitol, and glucose were selected as they are potentially capable to change water polarity; xanthan, PVP and microcrystalline cellulose were chosen because of their long chain molecular structure; citric acid, sodium citrate and baking soda were selected since they are either acidic or basic, all of which may improve the solubility of ellagic acid in water. However, the results showed no significant improvement for the solubility of ellagic acid in the extraction solution, except for baking soda, therefore the extraction method with the sodium bicarbonate assistance was further investigated.The SBAE extract with optimal parameters of RSM assay achieved an ellagic acid yield of 6.30 ± 0.92 mg/g from the raspberry wine pomace with an antioxidant activity of 79.0 ± 0.96 μmol Trolox eq/g pomace (DPPH assay). To our knowledge, this is the highest free ellagic acid yield as a result of extracts from raspberry-based materials so far. In previous studies, Vrhovsek et al.  has extrThe present study utilized raspberry wine pomace as the raw material for extraction, which is unprecedented to our knowledge. To profile the composition of ellagic acid pool, i.e., free ellagic acid, ellagic acid-conjugates and ellagitannins, it would be better to profile the extracted compounds with HPLC and consider the extracts from extraction methods with organic solvents of low acid/alkaline strength, since highly acidic or basic environments, especially when combined with elevated temperatures, could easily induce ellagitannins to hydrolyze ,42. Usin3 as the principal functional chemical, which may induce saponification of raspberry lipids and therefore improve the extraction performance. Clearly, saponification is greatly correlated with the temperature [3-induced saponification [Since ellagic acid and llagitannins compounds are actually embedded in a complex matrix of insoluble lipids, fibers and proteins within the seeds ,5, it woperature . As showfication , further3-induced saponification during the extraction process. Therefore, temperature and NaHCO3 concentration were of great importance, since they affect the lipid saponification process considerably.Similar tendencies have also been observed in studies concerning alkaline or alkaline enzyme extraction. In a recent study, Ayoub et al.  reported3 might cause degradation of ellagic acid, which was suggested by the reduction in ellagic acid yield from 1–2% NaHCO3 treatment to that of 3–4% NaHCO3  as the main alkaline reagent, which is less strong than that used in previous reports, and no organic solvent or protein enzyme is required, which further controls contamination by organic reagent and cut down the cost on reagents. Under the optimal conditions of 100 °C, 1.2% NaHCO3, 22 min and an S/L ratio of 1:93, an ellagic acid yield of 6.30 ± 0.92 mg/g was achieved from the raspberry wine pomace with an antioxidant activity of 79.0 ± 0.96 μmol Trolox eq/g (DPPH assay), both comparable to those in previous researches [Organic solvent extraction associated with various acidity and alkalinity levels have been widely applied to extract bioactive compounds in raspberry. For instance, Wang et al.  used aciproducts ,36,37. Hsearches ,36,38. TRaspberry wine pomace and wine samples were kindly offered by Liaoning Huayuan Winery Company . The pomace was collected after wine making (≈1 month), dried with outdoor air for 5 days and kept indoors at room temperature for 6 months. The material was a dry solid and a dark pink color, mainly consisting of raspberry seeds. Prior to the extraction, the pomace was ball-ground into a fine powder with a SCIENTZ-48 High-throughput Tissue Grinder  for 5 min at 50 Hz, and the fine powder was then stored at −20 °C before it was taken out for experiments.3, FeSO4 and NaHCO3 were of analytical grade and all bought from Shanghai Aladdin. Food additives of xanthan, polyvinyl pyrrolidone (PVP), maltodextrin, xylitol, citric acid, sodium citrate, glucose, microcrystalline cellulose and baking soda (sodium bicarbonate) were of food grade with >98% purity and purchased from Youbaojia Food Additive Co., Ltd. .Acetone, methanol and acetic acid solvents were all of analytical grade and purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. . Methanol and formic acid used for HPLC were of chromatographic grade and obtained from Spectrum Chemical Manufacturing Corp-China . Chemical reagents of ellagic acid, gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox, 2,4,6-tripyridin-2-yl-1,3,5-triazine (TPTZ), FeClv/v/v) method (MAW) as described by Ayoub et al. [v/v/v) solvent with a solid/liquid (S/L) ratio of 1:15 and then ultrasonicated at 30 °C for 20 min. The solution containing the extracted materials was obtained after centrifugation and the residues were subject to two more extractions in accordance with the first one. Three solutions obtained were joined together and studied for ellagic acid concentration with HPLC. Another set of ellagic acid was extracted by acetone–acetic acid–water  method (AAW) following the procedure described by Sojka et al. [Ground raspberry pomace samples were treated with organic solvents by procedures in two previous reports. Firstly, ellagic acid were extracted with methanol–acetone–water 3 concentration assay, 1 g of milled raspberry pomace was extracted by 0.1%, 0.5%, 1%, 2%, 3% NaHCO3 water solution, respectively, in an S/L ratio of 1:10 at 60 °C for 1 h shaking (200 rpm).For the NaHCO(2)3 solution in an S/L ratio of 1:10 for 1 h shaking (200 rpm).For the temperature assay, 1 g of milled raspberry pomace was extracted at 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C, respectively, with 1% NaHCO(3)3 solution at 100 °C in an S/L ratio of 1:10 S/L ratio.For the time assay, 1 g of milled raspberry pomace was extracted for 5, 10, 15, 20, 25 and 30 min, respectively, with 1% NaHCO(4)3 solution at 100 °C for 20 min.For the S/L ratio assay, 1 g of milled raspberry pomace was extracted in S/L ratios of 1:10, 1:50, 1:100, 1:150, and 1:200, respectively, with 1% NaHCOg for 5 min, followed by the supernatant undergoing subsequent ellagic acid quantification by HPLC analysis.For each assay, extraction was conducted in triplicates, and the mixture after extraction was centrifuged at 8000× 3 concentration, time and S/L ratio, were recognized as key extraction factors and selected as the independent variables  in the RSM assay, while an extraction temperature of 100 °C was set as a constant since it demonstrated optimal outcome compared to other temperatures in the above assay. A three-variable, three-level RSM assay  was designed by a Box–Behnken design, which required 17 extraction experiments as illustrated in b0 is the offset term, ib is the linear effect, iib is the squared effect, ijb is the interaction effect and iX and jX are independent variables. Model adequacy was evaluated by an F test and R2 calculations. The optimal parameters predicted by the model were thereafter performed in triplicates, and the actual data obtained were then compared to the predictions.The RSM assay was performed with the assistance of Design Expert Software . Three parameters, namely NaHCO4 as the standard. The DPPH radical scavenging activity was expressed as micromoles of Trolox equivalents per gram of milled raspberry pomace (μmol Trolox eq/g). The FRAP activity was expressed as millimoles of ferrous sulfate equivalents per gram of milled raspberry pomace (mmol Fe(II)S eq/g). Each assay was carried out in triplicates.Ellagic acid extracts obtained by MAW and AAW methods, as well as those retrieved with optimal parameters in the SBAE approach, were looked into their antioxidant activity by a DPPH method described by Brand-Williams et al. , and a fHPLC analysis were carried out using a Shimadzu LC-16 system , which consists of binary pumps, a UV detector, a ZORBAX SB-C18  column  and a LabSolutions Essentia control system . The flow rate was 1 mL/min and the injection volume was 20 μL. The monitoring wavelength for ellagic acid was 254 nm, and the mobile phase consisted of 0.3% formic acid in water (solvent A) and pure methanol (solvent B). HPLC separation was carried out by the following gradient conditions: 0–4 min 20–40% B, 4–13 min 40–90% B, 13–14 min 90–20% B, 14–16 min 20% B, with a total run time of 16 min. A standard curve to quantify ellagic acid was constructed in the range of 10, 25, 50, 100, and 200 mg/L. Sample peaks were identified based on their retention time compared with the ellagic acid standard.p < 0.05.Data were statistically examined by one-way analysis of variance (ANOVA) with R software  . Results3 concentration, time and S/L ratio were the key parameters for SBAE. By an RSM assay, the optimal SBAE conditions were established with the ellagic acid yield being 6.30 ± 0.92 mg/g and the antioxidant activity being of 79.0 ± 0.96 μmol Trolox eq/g (DPPH assay). The present SBAE method requires neither organic solvent nor protease or strong acid/alkaline reagents, whereas it achieved a higher extraction rate of ellagic acid than the conventional methods. The raspberry wine pomace was rich in free ellagic acid. The NaHCO3-induced lipid saponification and alkaline hydrolysis of ellagitannins could be the main reasons behind the improvement of ellagic acid extraction efficiency. Feasible procedure, good yield and reliable antioxidant activity of the extracts suggest the SBAE method to be useful for the application of ellagic acid extraction in the food and nutraceutical industry.A novel method named as SBAE was developed for ellagic acid extraction from the raspberry wine pomace using sodium bicarbonate as the main functional reagent. Extraction temperature, NaHCO


Estrogen receptors (ERs) regulate multiple complex physiological processes in humans. Abnormal ER signaling may result in various disorders, including reproductive system-related disorders , bone-related abnormalities, lung cancer, cardiovascular disease, gastrointestinal disease, urogenital tract disease, neurodegenerative disorders, and cutaneous melanoma. ER alpha (ERα), ER beta (ERβ), and novel G-protein-coupled estrogen receptor 1 (GPER1) have been identified as the most prominent ERs. This review provides an overview of ERα, ERβ, and GPER1, as well as their functions in health and disease. Furthermore, the potential clinical applications and challenges are discussed. Estrogen, a steroid compound, is primarily produced by the ovaries and placenta in females . The adrEstrogen, a lipid-soluble steroid hormone, is one of the most important female sex hormones. It is predominantly produced by the ovaries, testes, and adrenal cortex, and performs various crucial physiological functions. Estrogens are divided into two major categories, endogenous and exogenous, based on their origin. Endogenous estrogens are secreted by glands or cells in the body of living organisms, including estrogen in animals and phytoestrogens  in plants . In contTo date, four estrogens, estrone E1), 17β-estradiol (E2), estriol (E3), and estetrol (E4), have been identified in humans . The ter, 17β-estThe most common exogenous estrogens contain those metabolized and excreted by living organisms into the environment, selective estrogen receptor modulators (SERMs) targeting the ER for the treatment of endocrine disorders , and environmental pollutants generated by industrial and agricultural activities (such as polycyclic aromatic hydrocarbons and phenolic compounds) , 9. The Estrogens additionally play crucial roles in regulating the cardiovascular system, liver, pancreas, bone, brain, and immune system . EstrogeElwood Jensen first established the existence of ERs in 1958 by demonstrating that female reproductive tissue can take up estrogen by binding to proteins . In 1985In 2012, a new G protein-coupled membrane receptor (mER), GPER1, was identified using molecular cloning techniques . The hisESR1 on chromosome 6 (6q25.1) (ESR2 on chromosome 14 (14q23.2)   and ESR214q23.2) , respect.1  and E14q23.2) .Several isoforms of ERα have been identified arising from alternative gene splicing, including ERα-46  and ERα-GPER1 is encoded on chromosome 7 (7p22.3) and consists of 375 amino acids, with a molecular mass of 41 kDa . As a tyERα, ERβ, and GPER1 are the three predominant ERs. ERα is primarily expressed in the reproductive tissues , bone, white adipose tissue, kidney, liver, and breast. In contrast, ERβ is expressed in male reproductive organs, central nervous system (CNS), cardiovascular system, lung, immune system, colon, and kidney . GPER1 i2+ mobilization, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK). This process does not rely on gene regulation and occurs instantaneously within a time span of seconds to minutes; thus, it is referred to as a rapid “non-genomic effect”.Estrogens  bind to the traditional ERs (ERα and ERβ) and the novel receptor GPER1, exerting their genomic and non-genomic effects Figure 5ESR1 is highest expressed in the B cell, and intermediately expressed in CD4+ T cells, CD8+ T cells, NK cells, and plasmacytoid dendritic cell. Monocytes have the lowest levels of ESR1 RNA. Interestingly, ESR1 expression level increase in monocyte-derived dendritic cells, suggesting that ESR1 is induced during dendritic cell differentiation. ESR2 have highest level in B cells and plasmacytoid dendritic cells, and lower levels in other cell types. GPER1 are also expressed in various immune cells like T cell, B cell, mononuclear, macrophage, and neutrophils , and human epidermal growth factor receptor 2 (HER2) : luminalERα is overexpressed in breast cancer, which is approximately 10% in healthy tissues compared to 50–80% in breast cancer tissues . AdditioIn vitro studies revealed that the re-expression of ERβ in breast cancer cells repressed cell proliferation, promoted cell apoptosis, and sensitized the tumor cells to chemotherapy  and aromatase (CYP19A1)  , 175. AnCYP19A1) . These rCYP19A1) . DecreasCYP19A1) . FurtherCYP19A1) . In contCYP19A1) . CytoplaCYP19A1) . High nuCYP19A1) .via mERs. GPER1 expression is reportedly higher in ovarian endometriosis than in the normal endometrium, and this expression is associated with estrogen levels and the extent of inflammation  progression remains unclear. Serum estrogen levels are associated with the risk of prostate cancer development. This indicates that estrogen and ERs, which mediate the action of estrogens, may be risk factors , 179.via growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and IGF-1  are a collective group of disorders caused by abnormalities in the heart and blood vessels. They are the principal cause of death in both men and women. Epidemiologic studies have shown that prior to menopause, women are at a lower risk of developing CVD compared to men. Women also have decreased CVD-associated morbidity compared to age-matched men, and present with CVD 10 years later than men . Heart fMyocardial I/R injury and heart failure are two common heart-related diseases . I/R inj2+ accumulation compared to control mice  opening . AnotherIn conclusion, ERα, ERβ, and GPER1 contribute to myocardial protection following I/R injury Figure 6via MAPK and Akt signaling and enhances vascular remodeling (Hypertension (HTN), pulmonary hypertension (PH), and atherosclerosis are typical vascular diseases , whereinmodeling . ERα-knomodeling . ERα expmodeling . Loss ofmodeling . ERα tenmodeling . ERα alsmodeling  and decrmodeling .2O2 , EGFR, PI3K, and ERK  and esophageal cancer (EC) are the primary esophageal diseases. GERD is a recurrent disease characterized by abnormal reflux at the gastroesophageal junction. EC, one of the most common malignant tumors in the gastrointestinal tract, is divided into two histological subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). EAC can arise from chronic GERD . The preERα and ERβ are highly expressed in ESCC cell lines. However, the expression level and role of ERα in ESCC remain controversial. Nozoe et al. demonstrated that ERα is expressed in the cytoplasm of 64.4% tumor tissue in immunohistochemistry analysis . Dong etThe role of ERβ in esophageal diseases also remains controversial. Wang et al. suggested that estrogen in the areas of EC high incidence is relatively lower, and the inhibition role of estrogen against tumor is mediated by the ERβ . Nozoe eFew studies have focused on GPER1 in esophageal diseases. One study revealed that GPER1 overexpression and upregulation of the cancer suppressor gene beclin-1 in ESCC is associated with poor prognosis . The autTaken together, ERα and ERβ exhibits both inhibit and promote role in esophageal diseases Figure 6Peptic ulcers (PUs) and gastric cancer (GCs) are the most common gastric diseases. PU results in gastric and duodenal ulcers with complications such as upper gastrointestinal bleeding and gastric outlet obstruction. PU is relatively rare during pregnancy, suggesting that estrogen may exert a protective effect. The risk of developing PUs is lower in women than in men , 229. WoERs play pivotal roles in the occurrence and development of GC. Although ERα and ERβ are expressed during GC, ERβ, but not ERα, is abundantly expressed in GCs . Some stThe expression level of ERβ in tumor tissue is lower than in healthy tissue , 245. ERGPER1 expression levels are reportedly downregulated in GC tissues. The lower GPER1 expression tends to be negatively correlated with the survival of patients with GC . It alsoIrritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colon cancer (CC) are three major intestinal disorders. IBS is characterized by disorderly bowel movements and chronic abdominal pain. Some studies have highlighted the significant roles of estrogen and ERs in the pathogenesis of IBS. IBS  and CC  are moreERα expression is higher in IBS , and ERαThe expression of ERα-36 and ERα-46 in CC is higher than that in normal tissue. ERα-36 is associated with tumor stage and lymph node metastasis , suggestERα and GPER1 tend to show harmful effects, while ERβ shows a protecting role in intestinal diseases Figure 6The liver is a hormone-sensitive organ, and its functions are influenced by estrogens . Common In health liver tissue, men have higher ERα expression level than women, while no significant differences in ERβ expression. In addition, ERα:ERβ is higher in health men . ERα andERβ has been reported to be related to lipid metabolism. ERβ activation by genistein  improves hepatic lipid metabolism . ERβ is The role of GPER1 in the liver is complex. The expression of GPER1 in HCC tissues is lower than that in non-HCC tissues . The devTaken together, ERβ and GPER1 show inhibiting role in the progress of liver cancer through reducing the fibrosis and immune response, while the role of ERα remains controversial Figure 6The pancreas is an essential digestive organ in humans. The pancreas has endocrine and exocrine functions, mainly related to glucose metabolism. The external secretion of the pancreas is pancreatic juice, which is secreted from the pancreas and produces digestive enzymes that help digest fat. Islet cells produce insulin, which lowers blood sugar, and glucagon, which increases blood sugar. Pancreatic diseases include inflammatory diseases, pancreatic cysts, and other malignancies. When pancreatitis occurs, fat in the body is difficult to digest. Estrogens are highly associated with glucose metabolism . FurtherERα is significantly expressed in pancreatic cancer tissues and is related to tumor size, distant metastasis, and poor prognosis of pancreatic cancer. ERα promotes cell proliferation, tumor growth, migration, and invasion by activating epithelial-mesenchymal transformation . ERα knoHigh GPER1 expression in PDAC is associated with improved survival . AdminisCollectively, ERα and ERβ show promoting role in pancreatic disease and may be the therapy target Figure 6Escherichia coli. UCB is the most common bladder malignancy. Endogenous estrogen protects against UTIs (Urinary tract infections (UTIs) and urothelial carcinoma of the bladder (UCB) are the most common urogenital diseases. UTIs are bacterial infections, wherein 80% are caused by uropathogenic nst UTIs –273. Posnst UTIs . Many otnst UTIs , 276. InIn UTI, ERα is related to the bacterial clearance functions. In ovariectomized mice, ERα agonist PPT treatment decreases the bacterial load in the kidney, whereas treatment with the ERα antagonist MPP (methyl-piperidino-pyrazole) impairs bacterial clearance. These results suggest that bacterial clearance in the kidney is mediated by ERα . EstrogeUrothelial cells also express high GPER1 levels . A studyIn conclusion, both ERα and ERβ stimulates urothelial cell proliferation and tumor cells growth, while GPER1 inhibit urothelial cell proliferation Figure 6Neurodegenerative diseases are characterized by decreased mitochondrial activity, oxidative phosphorylation, and increased ROS production in the CNS. Common neurodegenerative diseases include Alzheimer’s disease (AD) and Parkinson’s disease (PD). AD is a progressive neurodegenerative pathology characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles, leading to neuronal dysfunction and cell death, negatively affecting cognition and memory in patients with AD . Postmen2+ signaling plays a vital role in maintaining normal brain function. ERα- and ERβ-selective agonists can effectively activate rapid intracellular Ca2+ influx in neurons, leading to downstream MAPK signaling and ERK phosphorylation, thus playing a neuroprotective role  phosphorylation (GPER1 controls melanin production and is expressed in melanoma cells of the skin . Specifirylation . GPER1 arylation . Furtherrylation . GPER1 srylation .In conclusion, ERα and ERβ exhibits pro-proliferative effects in melanoma by activating the downstream pathway like Akt and MAPK signaling pathways Figure 6Estrogens and their roles in ER signal transduction have become pivotal in developing various diseases, thereby making them potential therapeutic targets. ERs are expressed in various organs and regulate vital functions . We summBL and LOY data curation; PC writing original draft preparation; PC and BL writing—review and editing; LOY Supervision; PC visualization; All authors have read and agreed to the published version of the manuscript.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.


Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79–82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel. Among apathways .2+ absorption in the intestine and reabsorption in the kidney [2+ transport; in the uterus, with a potential role in establishing and maintaining pregnancy; and in exocrine organs such as the pancreas, prostate, mammary, salivary, and sweat glands [trpv6−/− mice demonstrated that TRPV6 serves as a principle mechanism for apical intestinal Ca2+ absorption [trpv6−/− mice exhibit disordered Ca2+ homeostasis, including defective intestinal Ca2+ absorption, increased urinary Ca2+ excretion, deficient weight gain, and reduced fertility, suggesting the pivotal role in calcium homeostasis in tissues where this channel is expressed.TRPV6 acts as the first step of the transcellular pathway, which is involved in many processes such as Cae kidney ,5. TRPV6t glands ,4. Studisorption . The trpThough no distinct isoform for this protein is known so far, some alternative translation of this protein has already been reported  and may It has already been demonstrated that the overexpression of the TRPV6 calcium channel is a common event in cancers of epithelial origin. TRPV6 appears to be up-regulated in tissue samples from ovary, prostate, breast, thyroid, colon, and pancreatic tumors . DespiteAmong them, the most important applications are the use of the TRPV6 channel as a diagnostic/prognostic marker. Intriguingly, one of the outstanding examples is prostate cancer (PCa) where TRPV6 is absent in healthy prostate and it becomes expressed in prostate adenocarcinoma and TRPV6 expression correlates significantly with the Gleason score, being significantly expressed in lymph node metastasis of prostate origin . Though https://www.uniprot.org/uniprot/Q9H1D0) has been analyzed, and four unique epitopes covering distinct domains, such as the N-terminus, X-loop, pore, and C-terminus, were designed to generate rabbit polyclonal antibodies , the extracellular loop (X-loop) (rb79), the pore region (rb82), and the C-terminus (rb81) and were subjected to further analysis.Four generated antibodies were firstly examined using SDS-PAGE in denaturing conditions . In our The immunoblotting of total lysates from LNCaP, DU-145, PC-3, and PC-3M cells revealed with rabbit polyclonal anti-TRPV6 antibody rb79 showed an expected size of the glycosylated form of the protein around 95–100 kDa, while the theoretical size of the unglycosylated protein is around 87.3 kDa A. In addThe immunoblotting of the same total lysates revealed with rabbit polyclonal anti-TRPV6 antibody rb80 of prostate cancer cells showed a high molecular band of approximately 160 kDa, which was only detected in LNCaP cells B. In LNCFinally, a rb82 antibody showed a staining of one band of 80 kDa exclusively in LNCaP cells, which is below the expected theoretical unglycosylated protein of approximately 87.3 kDa D.According to the obtained data, all antibodies except for rb79 were discarded since they did not show the expected size of the monomeric protein or the quality of detection.To pursue the antibody validation, four siRNAs were used in our studies to carry out a specific knockdown of the TRPV6 channel. The list of the siRNA sequences is indicated in As a next step, we used an overexpression system. It should be noted that it is extremely difficult to have a cell system in vitro that does not express the TRPV6 channel since the presence of 2 mM of calcium in almost every medium makes the expression of TRPV6 advantageous for the cell’s survival. Our data show an increase varying from slight to strong in TRPV6 expression C, suggestrpv6−/− cell line the expression of the TRPV6 channel was extinct. Surprisingly, a faint band was still present at the expected size of the TRPV6 channel , but alFinally, an immunoprecipitation using the rb79 antibody has been performed to confirm the use of this antibody as well as to see whether one can enrich the quantity of TRPV6. As shown in trpv6 gene is situated on the seven chromosome, locus 33–34, and consists of 15 exons  is shown as % in the right corner. In our experiment, we used a control plasmid and the other calcium channel, Orai1, fused to the YFP protein. As it can be seen from Of four generated antibodies, only rabbit polyclonal anti-TRPV6 antibody rb79 produced a stable and clear staining of prostate cancerous epithelial cells. This antibody has been used to perform IHC using 21 human clinical samples from prostate resection specimens . Six castrpv6−/− or HAP-1trpv6+/+ cell lines  against an epitope of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel around 100 kDa since this channel contains a strong putative N-glycosylation site as its close analog ,21. In o and PCR ,12,16,17In conclusion, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders where TRPV6 channels are involved.Human PC3M (metastatic cell line issued from PC3 cellules grafted in vivo), PC-3, LNCaP, DU-145, HEK293, and CHO-K1 cell lines were from American Type Culture Collection (ATCC) and were cultured in RPMI , DMEM (HEK293), and F12 (CHO) media  supplemented with 10% fetal calf serum, containing kanamycin (100 µg/mL) and L-glutamine (2 mM) where necessary. The HAP-1 cell line is a near-haploid human cell line that was derived from the male chronic myelogenous leukemia (CML) cell line KBM-7  and cul2 in the air. The medium was changed three times a week, and cultures were split by treating the cells with 0.25% trypsin (in PBS) for 5 min at 37 °C before reaching confluence. For the experiments, cells were seeded in 6-well plates for PCR and Western blotting. To maintain the −/−trpv6 status of the cells, the antibiotic selection G418 was used at a concentration of 200 µg/mL for the maintenance in culture of the HAP-1trpv6−/− cells.All the cells were cultured at 37 °C in a humidified atmosphere with 5% COg at 4 °C for 20 min, mixed with a sample buffer containing (denaturing conditions): 125 mM Tris-HCl pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, 0.01% Bromophenol Blue, and boiled for 5 min at 95 °C. For the non-denaturing conditions, both SDS and β-mercaptoethanol were avoided, and samples were not boiled. Total protein samples (50 µg) were subjected to 8% SDS-PAGE and transferred to a nitrocellulose membrane by semi-dry Western blotting . The membrane was blocked in a 5% milk containing TNT buffer  overnight then probed using specific rabbit polyclonal anti-TRPV6 antibodies  in a final volume of 25 µL. DNA amplification conditions included the initial denaturation step of 7 min at 95 °C and 40 cycles of 30 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and finally 7 min at 72 °C. The primers used are listed in RT-PCR experiments were performed as previously described . Total The quantitative real-time PCR of TRPV6 and HPRT mRNA transcripts was performed using MESA GREEN qPCR MasterMix Plus for SYBR Assay  on the Bio-Rad CFX96 Real-Time PCR Detection System. The sequences of primers are indicated in LNCaP cells were transfected with 40 nM of siRNA against TRPV6 (1–4 or mix) or siLuciferase  using 5 µL of Lipofectamine 3000 transfection reagent  following the manufacturer’s instructions  according to the manufacturer’s instructions. Briefly, 2 µg of the plasmid was transfected into 2 million trypsinized cells, which were then plated onto six-well dishes, 35 mm dishes, or onto the glass coverslips for 48 h.® 488 goat anti-rabbit IgG  was used as a secondary antibody for TRPV6 staining. Fluorescence analysis was carried out using a Carl Zeiss Laser Scanning Systems LSM 510 connected to a Zeiss Axiovert 200 M with a 63 × 1.4 numerical aperture oil immersion lens at room temperature. Both channels were excited, collected separately, and then merged using the Carl Zeiss LSM Image Examiner software (3.1.0.99).LNCaP cells grown on the glass coverslips were washed with PBS and immediately fixed in 4% paraformaldehyde in PBS. PBS-glycine (30 mM) was used to quench the reaction with the subsequent permeabilization with 0.1% Triton X-100. The cells were washed again in PBS and subjected to a conventional immunostaining procedure. Alexa Fluor2 cell culture flask were washed with PBS and immediately lysed with an ice-cold lysis buffer containing: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl, 1 mM PMSF, 1% Nonidet P-40, and a protease inhibitor cocktail from Sigma. Lysates were incubated with the lysis buffer on the rotating platform for 30 min at +4 °C, then transferred into the 15 mL tube and ultrasonicated for 10 s. The samples were then centrifuged at 15,000× g at 4 °C for 20 min, and the total protein concentration was measured. A small amount of the total lysates (50 µg) was kept for the input control, and an equal amount of total protein lysates among different conditions was added to the agarose beads prepared as follows: 50 µL of Agarose-Protein A/G conjugates beads were washed 3 times with the standard PBS pH = 7.4 solution, then the beads were incubated for 2 h at room temperature with 10 µg of the antibody, followed by the 3 washes with PBS to get rid of the unbound antibodies. The rb79rich protocol required a longer incubation time and a larger bead volume (100 µL) coupled to rb79. Antibody-Agarose-beads-protein A/G complexes were incubated overnight at +4 °C and washed the next day 3 times with the ice-cold PBS followed by 100 µL of 2× a sample buffer containing: 125 mM Tris-HCl, pH 6.8, 4% SDS, 5% ß-mercaptoethanol, 20% glycerol, 0.01% Bromophenol Blue, and then boiled for 5 min at 95 °C prior to the SDS-PAGE procedure described above.T47D cells grown in the 75 cm2 flasks as originally described [Flow cytometry assays were performed on cell populations cultured in triplicate in 25 cmescribed . ApproxiParaffinized human prostate anonymous tissue sections from 21 prostatectomies were obtained from the Department of Cell Pathology, Hôpital St. Vincent de Lille. Paraffin-embedded prostate tissue sections were subjected to conventional deparaffinization followed by antigen retrieval using citrate buffer at 95 °C in a water bath. After saturation in the solution containing 1% BSA and 0.05% Triton X100 in PBS-gelatin, the prostate sections were incubated with the specific antibodies, such as rabbit polyclonal anti-TRPV6 antibodies , overnight at 4 °C . Donkey The whole TRPV6 cDNA containing the 5′-UTR on the pCAGGS vector was provided by Dr. Ulrich Wissenbach from the Universität des Saarlandes, Germany. This sequence was used to obtain a final vEF1ap-5′UTR-TRPV6_CMVp-mCherry vector , which was nucleofected into the cells, and the transfection rate was evaluated using a control vEF1ap-5′UTR_CMVp-mCherry vector. pTRPV6-eYFP and pOrai1-YFP vectors were used as previously described .trpv6−/− cell model has been created using the HAP-1 cell line, which is a near-haploid human cell line that was derived from the male chronic myelogenous leukemia (CML) cell line KBM-7 [The HAP-1ne KBM-7 . A knockv/v with glycerol and stored at −20 °C.The 15 amino acid antigens were synthesized, coupled to a KLH protein at its N-terminus, and injected into the rabbits once per week for four weeks following the final bleed . The serum was tested by ELISA using antigen-coated plates, followed by affinity purification in columns against the same bound antigen. Final affinity-purified antibodies were obtained and diluted 50/50 All reagents were purchased from Sigma  unless otherwise specified.p < 0.05 was considered significant. In the graphs, (*) and (**) denote statistically significant differences with p < 0.05 and p < 0.01, respectively.For each type of experiment where the data were analyzed using statistics, the data were accumulated from at least three independent experiments. Data were analyzed using Origin 7.0  software. Results were expressed as Mean ± S.E.M., where appropriate. N equals the number of series of experiments and n equals the number of cells used in the study. ANOVA was used for the statistical comparison of the differences, and This work is an integral part of patent EPO, number EP21306438. Title: “ANTIBODIES AGAINST EXTRACELLULAR EPITOPES OF HUMAN TRPV6 CHANNEL AND THEIR DIAGNOSTIC AND THERAPEUTIC USES”, inventors: Dr. V’yacheslav Lehen’kyi, Dr. Aurélien Haustrate, and Prof. Natalia Prevarskaya, filed on 14 October 2021. .


TP53 gene knockdown provides new data for understanding the limitations of HaCaT cells when using them as an experimental model of normal human keratinocytes.The HaCaT line of immortalized non-tumor cells is a popular model of keratinocytes used for dermatological studies, in the practice of toxicological tests, and in the study of skin allergic reactions. These cells maintain a stable keratinocyte phenotype, do not require specific growth factors during cultivation, and respond to keratinocyte differentiation stimuli. HaCaT cells bear two mutant p53 alleles - R282Q and H179Y. At least two mechanisms of GOF (gain-of-function) of mutant p53 are known: it affects functions of p63/p73 by inhibiting their binding to DNA; or it binds to new DNA sites by interacting with other transcription factors . Proteins of the P53 family play an important role in the regulation of proliferation and differentiation processes of human keratinocytes. Proteomic study of HaCaT cells with In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of human immortalized HaCaT keratinocytes and p53 knockdown HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS/MS measurements followed by their processing with MaxQuant software (version 1.6.3.4). The “RAW” files were deposited to the ProteomeXchange with identifier PXD033538. Dataset covers 10 biological samples. Information about samples is presented in ELISA and Western Blot were employed for knockdown verification. Transduction with anti-TP53 shRNA led to a significant decrease of intracellular p53 concentration .Fig. 1p5Comparison of wild-type and p53 knockdown HaCaT protein profiles resulted in the identification of 2875 proteins, out of which 320 were significantly changed, including 222 and 98 proteins that were up- and downregulated in p53 knockdown HaCaT, respectively. The STRING analysis of differently expressed proteins for p53 knockdown HaCaT is presented in 22.1Sodium chloride, taurocholic acid sodium salt (TCA), and acetonitrile were provided by Merck (Germany), formic acid was from ACROS Organics (USA), tris-(2-carboxyethyl)-phosphine (ТСЕР), methanol, trifluoroacetic acid (TFA) were from Fluka (Germany). Modified trypsin was obtained from Promega (USA). For cell cultivation, we used DMEM/F:12 medium (1:1), GlutaMAX, fetal bovine serum, PBS, penicillin/streptomycin .2.2HaCaT cells were obtained from Bank of DKFZ, Heidelberg and cultured in DMEM/F:12 medium  supplemented with 1% GlutaMAX , penicillin/streptomycin  and 10% v/v fetal bovine serum . Cells were grown in 60 mm Petri dishes  and harvested by trypsinization. The culture medium was replaced by fresh every other day.2.3To knock down TP53 gene expression in HaCaT cell line lentiviral particles (LVPs) encoding anti-TP53 shRNA pLKO-p53-shRNA  were used. To assemble the LVPs HEK293T cells were transfected with pLKO-p53-shRNA vector using the standard PEI transfection protocol 2.44 cells per well 48 hours prior to analysis. Total cellular protein level was measured with a spectrophotometer. Samples were diluted with PBS to reach protein concentration of 200 µg/ml. Signal intensity was measured at 450 nm. Two biological replicates per group were used for ELISA. Difference between the groups was tested using Student's t-test. Difference was considered significant at p < 0.05.For semi-quantitative determination of p53 protein level, Abcam ELISA kit (ab205713) was used. Cells were seeded into a microtiter plate at a density of 1 × 102.56 of wild type and p53 knockdown cells were harvested by trypsinization for Western blot analysis. The cell pellet was lysed in RIPA buffer .Verification of knockdown was done using 8% sodium SDS-PAGE. 1 × 10The proteins were transferred onto the PVDF membrane following immunoblotting with anti-p53  and β-actin  antibodies as a loading control. Alkaline phosphatase-conjugated secondary antibodies  were used for primary antibodies recognition. The chemiluminescent reaction was detected with DNR LumiBis Gel Imaging System 3.2. The images were obtained using GelCapture software. Three biological replicates per group were used for Western Blot. Difference between the groups was tested using Student's t-test. Difference was considered significant at p < 0.05.2.6After reaching confluency of 75%, cells were washed three times with PBS and then harvested mechanically with cell scrapers , pelleted with centrifugation, and incubated in 100 µL lysis buffer  on an orbital shaker at room temperature for 20 min followed by a 5 min incubation at 95 °C.Cooled down samples were sonicated three times at room temperature using Bandelin 2070  for 20 seconds at 90% power.2O were added, shaken again on a vortex, and centrifuged at 14000g for 2 minutes. Next, the upper layer (aqueous) was removed, and methanol was added in a volume of 400 µl. The resulting solution was vortexed and centrifuged for 5 min at 14,000 g to precipitate proteins. Methanol was carefully removed, and the precipitate was dried in an Eppendorf Concentrator 5301  at 45 °C for 5 min.Proteins were precipitated using the methanol-chloroform method 2.7Pierce™ BCA protein assay kit  was used to determine the protein concentration. For this purpose, 20 µl of a denaturation buffer of the composition 5M urea, 1% TCA, 15% acetonitrile, 50 mM phosphate buffer, pH 6.3, 300 mM sodium chloride was added to 50 µg of protein extracts.Further, to recover the protein, a 25 mM TCEP solution in 0.1 M ammonium bicarbonate was added in a volume of 5 µl for each sample and incubated at room temperature for 45 min. After that, a solution of 300 mm IAA in 0.1 M ammonium bicarbonate was added in a volume of 5 µl, incubated for 30 minutes in the dark, and the remaining IAA was suppressed with 5 µl of 300 mM DTT solution in 0.1 M ammonium bicarbonate. The volume of the reaction mixture was adjusted to 200 µl with 0.1 M ammonium bicarbonate solution, and trypsin was added in the ratio enzyme:protein 1:50.2O, then the samples were loaded onto Zip-Tips and washed three times with 0.1% TFA and 5% methanol in ddH2O. The peptides were eluted with 70% acetonitrile with 0.1% formic acid. The elution of the peptides from the column was carried out with a solution of 0.1% formic acid in 70% acetonitrile.Samples were incubated overnight at 37 °C with rotation. Samples were centrifuged at 10 °C and 12,000 × g for 10 min, then the supernatants were collected and purified using a C18 ZipTip according to the manufacturer's protocol 2.8Separation of the peptides was carried out on an Acclaim µ pre-column . For this, a peptide solution (1 µg of peptide in 1-4 µl of 0.1% formic acid) was applied directly to the column. The concentration of peptides was performed at a flow rate of 10 µl/min in the isocratic mode of mobile phase C  for 4 min. To separate the peptides, a high-performance liquid chromatography system Ultimate 3000 Nano LC  was used. Separation was carried out on a C18 column . Elution was performed at a flow rate of 0.3 µl/min with a gradient of 0.1% formic acid (buffer A) and 80% acetonitrile, 0.1% formic acid (buffer B).Initially, the columns were equilibrated with buffer A for 12 min, and then, gradually (over 95 minutes), the concentration of buffer B was increased from 5 to 35%. Then for 6 minutes, the concentration of buffer B was increased to 99%. The columns were washed several times with 99% buffer B and equilibrated with buffer A for a7 minutes.TM  was used for MS analysis. Each sample was examined in three technical repeats. Analysis conditions: capillary temperature 240 °C, ionizing voltage 2.1 kV. Spectra were obtained in the range of 300-1500 m/z, resolution 120000 (MS). Tandem spectra were obtained in the range 140-2000 m/z, resolution 15000 (MS/MS). The maximum integration for precursor and fragment ions was 50 ms and 110 ms, respectively. AGC target was set to 1 × 106 and 2 × 105 for precursor and fragment ions, respectively. For the selection of the precursor, an isolation intensity threshold of 50,000 counts was determined. For fragmentation with high energy collisional dissociation at 29 NCE, up to 20 best precursors were selected. Precursors with a +1 and more than +5 charge state were excluded, and all measured precursors were dynamically rejected from triggering of a following MS/MS for 20 s.Mass spectrometer Q Exactive HF-X Hybrid Quadrupole-OrbitrapThe equipment of the “Human Proteome” Core Facility of the Institute of Biomedical Chemistry  was used for mass spectrometric measurements. Data from proteomic studies were submitted to the ProteomeXchange Consortium through the PRIDE 2.9MaxQuant software (version 1.6.3.4) was used for MS/MS spectra identification. Proteins were identified using Homo sapiens reference proteome UP000005640, contained 20 361 reviewed and 58 691 unreviewed entries. The database was downloaded in July, 2021.Following identification parameters were used for the database search: enzyme specificity - Trypsin, maximum cleavages allowed – 2, MS1 and MS2 tolerances – 5.0 ppm and 20 ppm, respectively, fixed modification - carbamidomethylation (Cys), variable modifications - N-terminal proteins acetylation and methionine oxidation (Met), false discovery rate estimated using the decoy hit distribution for Peptide Spectrum Matches (PSMs), peptides and proteins identification −1.0%.Daniil D. Romashin: methodology, investigation, resources, data curation, writing—original draft preparation, writing—review and editing; Alexander L. Rusanov: Conceptualization, methodology, investigation, resources, validation, writing—original draft preparation, writing—review and editing, project administration; Peter M. Kozhin: methodology, investigation, writing—original draft preparation, writing—review and editing; Maxim N. Karagyaur: methodology, investigation, writing—original draft preparation, writing—review and editing; Olga V. Tikhonova: investigation, writing—original draft preparation, writing—review and editing; Victor G. Zgoda: methodology, investigation, data curation, writing—original draft preparation, writing—review and editing; Nataliya G. Luzgina: Conceptualization, investigation, writing—original draft preparation, writing—review and editing, supervision, project administration.This work did not involve the use of human subjects or animal experiments.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.