Intestinal Protection By Proanthocyanidins Involves Anti‑oxidative And Anti‑infammatory Actions in Association With An Improvement Of Insulin Sensitivity, Lipid And Glucose Homeostasis Part 1

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The gastrointestinal (GI tract is a complex system that fulfills essential functions in the human body-3. It ensures digestion and absorption of nutrients, contributes to overall immunology competence by providing protection against pathogens, produces multiple peptidic hormones acting on several tissues and pathways, and harbors microbial communities interacting with the host and influencing metabolic health45. In addition, the intestinal epithelium protects from foreign pathogen invasion and serves as the first line of defense, which is crucial for the prevention of multiple disorders6.

The GI tract is continuously exposed to harmful stimuli that may cause oxidative stress(OCS), inflammation, and injury. Intraluminalpro-oxidants from ingested nutrients contain varying concentrations of lipid oxidation products, such as cholesterol oxides',8. The intestinal epithelium is also subject to multiple oxidant assaults from

Figure 1. Effects of PACs on cell integrity and viability of Caco2/15 cells. The integrity of the Caco 2/15 monolayer was determined by cell viability, differentiation, and tight junction assays in a fully differentiated state.

Proanthocyanidins(PACs,250 ug/mL) were added to the apical cell compartment for 24 h prior to treatment with Fe/Asc(200 uM/2 mM)for 6h at 37C. cistanche extract powder (A) MTT was used to assay cell viability, and (B) transepithelial resistance was also determined. (C) Villain, (D) Occludin, and (E) Claudin protein expressions were assessed by Western blot. Results represent the means±SEM of 3 independent experiments, each in triplicate. A representative Western blot is shown, illustrating an experiment in triplicate on the same gel and at the same exposure.

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catalase-negative bacteria, oxidase-producing desquamated cells (e.g, xanthine oxidase), and hypothiocvanous acid-containing saliva, which increase reactive oxygen species(ROS)in intestinal lumen'.Importantly, ROS are able to damage cell membranes, disturb barrier integrity, and lead to enhanced intestinal permeability, inflammation, and endotoxemia. The combination of OCS and inflammation in the gut compromises immune digestive, endocrine, and nervous functions. In particular, it elicits a decline in insulin sensitivity (IS), which triggers metabolic disordersl2,13.

Recently, growing evidence suggests that polyphenols and derivatives have the potential to prevent and treat chronic diseases'4. These plant secondary metabolites exhibit various biological features and are gaining a growing interest among scientists. They are capable of reducing ROS generation, neutralizing OCS development, and fighting inflammation5,16. cistanche genghis khan, However, the mechanisms of action resulting in beneficial outcomes remain elusive. Furthermore, given their limited absorption and bioavailability, investigations into their physiological functions were limited to polyphenol transport and catabolism in the gut.

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The main objective of the study is to determine the modulation of OxS and inflammation by proanthocyanidins (PACs), which are abundant flavan-3-ols in dietary fruits, vegetables, nuts, and grains. As OCS and inflammation are two crucial processes promoting IS, insulin signaling is evaluated in response to PACs. The impact of these polyphenols is also assessed on fatty acids(FA)β-oxidation and lipogenesis, cistanche in your pants which will allow estimating lipid homeostasis. The mechanisms behind PAC influence are appraised by focusing on central transcription factors. To this end, we have used the well-established human-derived immortalized Caco-2/15 cell line. These intestinal cells are known to undergo a spontaneous cell differentiation leading to the formation of a cell monolayer expressing several morphological and functional characteristics of mature human enterocytesi7-19.

Results

Caco 2/15 cell integrity following different treatments. Before evaluating the impact of polyphenols on various processes, we wanted to ensure that Caco-2/15 cells maintained their integrity in response to iron/ascorbate (Fe/Asc) and PACs treatment. Cell viability was not affected as observed from the invariable 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl Tetrazolium Bromide (MTT)assay (Fig. 1A). Confirmation of integrity preservation was obtained following analysis by trypan blue exclusion(data not shown). Furthermore, measurement of transepithelial electrical resistance did not disclose any significant modification of the barrier integrity

Figure 2. Effects of PACs on lipid peroxidation, endogenous antioxidants, and oxidative stress key transcription factors in Caco-2/15 cells. Proanthocyanidins （PACs，250 μg/mL） were added to the apical cell compartment for 24 h prior to treatment with Fe/Asc（200μM/2 mM） for 6h at 37℃C. Lipid peroxidation was determined by measuring (A)MDA levels by HPLC. The protein expression of (B)SOD2,(C) GPx,(D)NRF2, and (E)Keep 1 was determined by Western blot. (F)The ratio NRF2/Keapl was calculated. Results represent the means±SEM of 3 independent experiments, each in triplicate. A representative Western blot is shown, illustrating an experiment in triplicate on the same gel and with the same exposure.*P<0.05;***P<0.001 ys untreated cells(ctrl).*P<0.05;#P<0.01;#*P<0.001 vs Fe/Asc-treated cell.MDA malondialdehyde, SOD2 superoxide dismutase 2, GPx Glutathione peroxidase, NRF2 Nuclear factor erythroid 2-related factor 2, Keap1 Kelch-like ECH-associated protein.

Effects of PACs on Fe/Asc-induced OCS. cistanche near me We then estimated the induction of OCS following Fe/Asc administration. Assessment by HPLC of the malondialdehyde (MDA), as a biomarker of lipid peroxidation, showed an elevated MDA content compared with control Caco-2/15 cells(Fig. 2A). Consistently, the protein expression of the antioxidant superoxide dismutase 2(SOD2)and glutathione peroxidase(GPx) enzymes were reduced by Fe/Asc compared to untreated cells (Fig.2B, C). Nonetheless, the presence of PACs counteracted Fe/Asc-mediated lipid peroxidation as evidenced by MDA lessening as well as SOD2 and GPx elevation. As the transcription factor nuclear factor erythroid-2-related factor 2(NRF2)is critical for the regulation of endogenous antioxidants, we evaluated its protein expression(Fig.2D). No changes in NRF2 were detected in Caco-2/15 cells treated with Fe/Asc and PACs, but the protein expression of its negative regulator Kelch-like ECH-associated protein 1(Keap1), a biosensor for oxidative and electrophilic stresses, was upregulated by Fe/Asc and downregulated by PACs(Fig.2E).In particular, the important ratio of NRF2/Keap1, which is an indicator of transcriptional activation of NRF2, followed the same trend(Figs.2F). Overall, PACs are effective in counteracting Fe/Asc-mediated OCS.

PAC effects and mechanisms of actions with respect to the inflammatory process. Pro-inflammatory cytokines are triggered in intestinal epithelial cells in OCS conditions. Our experiments confirmed this observation by demonstrating a Fe/Asc-mediated tumor necrosis factor-alpha(TNFα)induction (Fig.3). In line with these data, the protein expression of cyclooxygenase-2(COX2), an enzyme exerting a multifaceted role in inflammation promotion and perpetuation, was increased by the pro-oxidant Fe/Asc. However,cistanche para que serve PAC administration offset TNFα and COX2 protein levels elicited by Fe/Asc (Fig. 3A, B). Since nuclear factor kappa-B(NF-KB)represents a powerful mediator of inflammatory responses, we tested its implication in the transcription of the aforementioned inflammatory components (Fig.3C). Whereas Caco-2/15 cells challenged by Fe/Asc exhibited a high NF-KB signal likely through activating NF-KB/IkB ratio (Fig.3E) and phosphorylation of its inhibitor

Figure 3. Effects of PACs on inflammatory markers and the key transcription factor NF-kB in Fe/Asc-induced inflammation in Caco-2/15 cells. Proanthocyanidins(PACs,250 ug/mL)were added to the apical cell compartment for 24 h prior to treatment with Fe/Asc(200 uM/2 mM)for 6 h at 37°C. Protein expressions of (A)COX2 and(B)TNFa,(C) NF-kB,(D)IkB, and its(F)phosphorylated form (pike)were determined by Western blot. The ratios(E) NF-kB/IkB and(G)plan/IkB were calculated. Results represent the means±SEM of 3 independent experiments, each in triplicate. A representative Western blot is shown, illustrating an experiment in triplicate on the same gel and with the same exposure.*P<0.05;**P<0.01 vs untreated cells(ctrl).P<0.05;P<0.01 vs Fe/Asc treated cells. COX2 Cyclooxygenase-2, TNFα Tumor necrosis factor-alpha, NF-xB nuclear transcription factor-kappa B, IkB Inhibitor of kappa B.

Regulation of intracellular lipid metabolism by PACs. The next step was to appraise the effect of PACs on lipid homeostasis by focusing on key proteins, that regulate FA β-oxidation and lipogenesis. We first examined the expression of carnitine palmitoyltransferase I(CPT-la) because this protein is the rate-controlling enzyme of the FA β-oxidation pathway. A lower level of protein expression characterized CPT-lα in response to Fe/Asc treatment, while the pre-incubation with PACs prevented its decline compared to untreated cells (Fig. 4A). These findings prompted us to determine the potential mechanisms of action by scrutinizing the protein mass of peroxisome proliferator-activated receptor alpha (PPARa) and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-la) as they represent two transcription factors necessary for the high-level expression of mitochondrial FA β-oxidation. Western blot analysis showed a slight downregulation of PPARa without changes in PGC-1a(Fig.4B, C). Nevertheless, the presence of PACs prevented Fe/Asc-mediated PGC-1a and PPAR-α decline. Thereafter, we turned toward the regulatory enzymes controlling the lipogenesis process. As shown in Fig.4, an important drop of phosphorylated acetyl CoA-carboxylase (ICC) and a marked rise of fatty acid synthase (FAS) were noticed following Fe/Asc administration, indicating the activation of lipo-genesis(Fig. 4D, E). These changes were probably due to the induction of the transcription factors PPARy and sterol regulatory element-binding protein lc (SREBP1c)in (aco-2/15 cells ( Fig,4EG)The presence of PACs was beneficial as it reversed the pro-lipogenesis effects of Fe/Asc. Since AMP-activated protein kinase alpha (AMPKa) represents a central regulator of FA β-oxidation and lipogenesis, we investigated its protein expression, which was found to be elevated in response to Fe/Asc while normalizing in response to PACs. On the whole, most of these findings sustain a protective role of PACs in lipid homeostasis (Fig.5).

Impact of PACs on intracellular glucose metabolism and insulin resistance. Gluconeogenesis is strictly controlled by the activities of rate-limiting enzymes such as phosphoenolpyruvate carboxykinase (PEPCK)and glucose-6-phosphatase(G6Pase). We explored the status of these enzymes by analyzing their pro-

Figure 4. Effects of PACs on FAβ-oxidation and lipogenesis in Caco-2/15 cells. Proanthocyanidins(PACs, 250μg/mL），cistanche penis were added to the apical cell compartment for 24 h prior to treatment with Fe/Asc （200 μM/2 mM）for 6 h at 37 ℃C. The protein expression of specific markers of FA β-oxidation:((A) CPTla,(B) PPARa, and (C) PGClα as well as of lipogenesis: (D) ACCphosphorylation,(E) FAS,(F) PPARy and(G) SREBP1) was assessed by Western-blot. Results represent the means± SEM of3 independent experiments, each in triplicate. A representative Western blot is shown, illustrating an experiment in triplicate on the same gel and at with same exposure.*P<0.05;**P<0.01 vs untreated cells (ctrl).*P<0.05;P<0.01 vs Fe/Asc treated cells. CPT carnitine palmitoyltransferase alpha, PPAR peroxisome proliferator-activated receptor, PGCIa peroxisome proliferator-activated receptor gamma coactivator 1-alpha, ACCAcetyl-CoA carboxylase, FAS Fatty acid synthase, SREBP1-c Sterol regulatory element-binding protein.

Figure 5. Effects of PACs on AMP-activated protein kinase alpha(AMPKa)regulating fatty acid β-oxidation and lipogenesis in Caco-2/15 cells. Proanthocyanidins (PACs,250 ug/mL) were added to the apical cell

compartment for 24h prior to treatment with Fe/Asc（200 μM/2 mM） for 6h at 37℃C. The protein expression of AMPKa was assessed by Western Blot. Results represent the means±SEM of 3 independent experiments, each in triplicate. A representative Western blot is shown, illustrating an experiment in triplicate on the same gel and with the same exposure.*P<0.05 vs untreated cells(ctrl). *P<0.05,**P<0.01 vs Fe/Asc treated cells.

This article is extracted from Scientifc Reports | (2021) 11:3878 | https://doi.org/10.1038/s41598-020-80587-5