Anti-inflammatory activity of high and low methoxylated apple pectins, in vivo and in vitro
AbstractOne of the possible mechanisms of the anti-inflammatory action of pectins is associated with the inhibition of excessive pro-inflammatory activity of macrophages - the cells that regulate inflammation intensity and reparative regeneration. It has been found that pectins with a low degree of methyl esterification of the carboxyl groups of the galacturonan core of the macromolecule exhibit this effect. In addition to leukocytes, intestinal epithelial cells are also involved in the pathogenesis of inflammatory bowel diseases. However, to date, there have been insufficient studies of the effect of pectin methyl esterification on the inflammatory response of enterocytes. The aim of the research was to evaluate the effect of the degree of pectin methyl esterification on inflammation of the colon in mice after oral administration and on the inflammatory response of human colon epithelium cells of the Caco-2 line in vitro.
Material and methods. In a prospective study, 40 male BALB/c mice weighing 20-25 g were used, 10 animals in each group. Solutions of apple pectins (200 mg/0.2 ml) were orally administered to mice through a plastic catheter 24 h before the induction of colitis. The control mice received water, and prednisone administration at a dose of 5 mg/kg of body weight was used as a positive control. Colon inflammation in mice was induced by a single rectal administration of 5% acetic acid (0.1 ml). A day later, the degree and area of the lesion was assessed using a light microscope, the activity of myeloperoxidase in the wall of the colon was determined by spectrophotometry. The effect of pectins on metabolic activity, intercellular permeability, Tumor Necrosis Factor a generation and alkaline phosphatase (ALP) activity in Caco-2 cells was assessed.
Results. It was found that low-methyl esterified pectin AU701, which contains more than 70% of free carboxyl groups, inhibited colon inflammation in mice. High methyletherified pectin AU201, in which more than 70% of the carboxyl groups are replaced by methyl ester, didn’t affect inflammation. It was revealed that pretreatment of Caco-2 cells with AU701 and AU201 pectins prevented lipopolysaccharide-induced increase in intercellular permeability and reduced the pro-inflammatory response of Caco-2 cells to LPS. After incubation of Caco-2 cells with AU701 pectin, the rate of hydrolysis of β-nitrophenylphosphate (an alkaline phosphate substrate) increased by 40%. Pectin AU201 had no effect on the alkaline phosphatase activity of enterocytes.
Conclusion. Thus, it was found that low-methyl esterified pectin AU701 inhibits inflammation both in vivo and in vitro. High-methyl esterified pectin AU201 suppresses pro-inflammatory reactions only in vitro. The ability of pectins to inhibit intestinal inflammation has a multifactorial nature, and is due, inter alia, to their ability to stimulate the expression of alkaline phosphatase by enterocytes.
Keywords:pectin, methyl esterification, inflammation, colon, Caco-2, alkaline phosphatase, tumor necrose factor-alpha, lipopolysaccharide, intercellular permeability
Funding. The work was carried out at the expense of subsidies for the implementation of a state task.
Conflict of interest. The authors declare no conflict of interest.
For citation: Markov P.A., Volkova M.V., Khasanshina Z.R., Martinson E.A., Popov S.V. Anti-inflammatory activity of high and low methoxylated apple pectins, in vivo and in vitro. Voprosy pitaniia [Problems of Nutrition]. 2021; 90 (6): 92-100. DOI: https://doi.org/10.33029/0042-8833-2021-90-6-92-100 (in Russian)
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Установлено, что через 1 ч после внесения ЛПС проницаемость монослоя клеток Caco-2 увеличивалась более чем в 4 раза. Повышенная межклеточная проницаемость сохранялась на всем протяжении периода наблюдения (рис. 4А). Предварительная обработка клеток Caco-2 пектинами AU701 и AU201 предотвращала ЛПС-индуцированное увеличение межклеточной проницаемости соответственно на 44±8 и 25±5% (рис. 4Б). Инкубация пектинов с клетками Сасо-2 не вызывала изменений в межклеточной проницаемости (см. рис. 4Б).
)
Рис. 4. Проницаемость клеточного монослоя клеток Сасо-2 для трипанового синего после внесения в среду ЛПС (А); влияние пектинов на ЛПС-индуцированную проницаемость монослоя клеток (Б) (M±σ, n=7)
Статистически значимое (p<0,05) отличие: * - по сравнению с предыдущей точкой измерения; а - по сравнению с контролем; б - по сравнению с AU201. Здесь и на рис. 5: ФБР - фосфатно-буферный раствор; ЛПС - липополисахарид; AU701 - высоко-, AU201 - низкометилэтерифицированный яблочный пектин.
Fig. 4. Permeability of the Caco-2 cell monolayer for trypan blue after adding LPS to the medium (A); effect of pectins on LPS-induced permeability of a cell monolayer (B) (M±σ, n=7)
Statistically significant (p<0.05) difference: * - compared with the previous measurement point; а - compared with the control; б - compared with AU201. Here and in Fig. 5: LPS - lipopolysaccharide; PBS - phosphate buffered saline, control; AU701 - high-, AU201 - low-methyl etherified apple pectin.
Через 1 сут после внесения ЛПС к клеткам Caco-2 концентрация ФНОα в культуральной среде увеличивалась более чем в 3 раза. Предварительная обработка клеток пектином AU701 снижала их ответ на ЛПС почти в 2 раза, в то время как обработка AU201 только на 30% (рис. 5).
)
Рис. 5. Влияние пектинов на генерацию клетками Сасо-2 фактора некроза опухоли а (ФНОа) до и после внесения липополисахарида в культуральную среду (M±a, n=7)
* - статистически значимое (p<0,05) отличие от контроля.
Fig. 5. Effect of pectins on the generation of tumor necrosis factor а (TNF-а) by Caco-2 cells before and after the introduction of lipopoly-saccharide into the culture medium (M±a, n=7)
* - statistically significant (p<0.05) difference from control.
Ранее было показано, что действие пектинов на ЛПС-стимулированный воспалительный ответ может быть обусловлено блокированием взаимодействия ЛПС c его рецептором [3]. Однако, по сообщениям ряда авторов, в качестве дополнительного противовоспалительного фактора также может выступать и ЩФ, локализованная на щеточной кайме энтероцитов. Известно, что ЩФ способна гидролизовать ЛПС и снижать тем самым его провоспалительный потенциал [25, 26].
Результаты проведенных нами экспериментов по характеристике влияния этерификации пектинов на активность ЩФ клеток Caco-2 показали, что пектин AU701 увеличивал удельную активность ЩФ. После инкубации клеток с пектином AU701 скорость гидролиза β-нитрофенилфосфата, субстрата ЩФ, увеличилась на 40%. Пектин AU201 не оказывал влияние на активность ЩФ энтероцитов человека (рис. 6). Следует отметить, что пектин AU701 оказывает влияние на активность ЩФ только в первые 48 ч совместной инкубации.
)
Рис. 6. Влияние пектинов на активность щелочной фосфатазы (ЩФ) в клетках Сасо-2 (M±a, n=7). Данные выражены в % к контролю в день измерения
* - статистически значимое (p<0,05) отличие от контроля.
Fig. 6. Effect of pectins on alkaline phosphatase activity in Caco-2 cells (M±a, n=7). Data are expressed as % of control
* - statistically significant (p<0.05) difference from control.
Таким образом, установлено, что низкометилэтерифицированный яблочный пектин защищает стенку толстой кишки от кислотно-индуцированного повреждения. Противовоспалительное действие пектинов реализуется не только через изменение функциональной активности лейкоцитов, как было показано ранее, но и через повышение устойчивости эпителиального барьера клеток кишки к повреждающим факторам.
Заключение
В проведенном исследовании выявлено, что низкоме-тилэтерифицированный яблочный пектин AU701 ингибирует воспаление как в условиях in vivo, так и in vitro. Высокометилэтерифицированный пектин AU201 подавляет провоспалительные реакции только в условиях in vitro. Полученные результаты указывают на то, что при пероральном поступлении пектинов в организм степень метилэтерификации карбоксильных остатков галактуроновой кислоты, при прочих равных структурных характеристиках пектинов, имеет решающее значение для их проявления противовоспалительного действия. Способность пектинов ингибировать кишечное воспаление имеет многофакторную природу и может быть обусловлено в том числе их способностью усиливать барьерную функцию клеток эпителия и стимулировать экспрессию щелочной фосфатазы клетками кишечника.
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