Study On The Effect And Mechanism Of Food Emulsifier Polysorbate 80 On Promoting Intestinal Absorption Of MEHP

BackgroundPhthalate esters(PAEs)are widely used as plasticizers in a variety of consumer products,including food packaging bags and toys,because they enhance the softness,elasticity and durability of plastic products.Among them,di(2-ethylhexy1)phthalate(DEHP)is the most widely used and the largest amount of PAEs.DEHP is a kind of recognized environmental endocrine disrupting chemicals(EEDCs).It is widely used in medical equipment,chemical and plastic products,and leach into the atmosphere,water,food and even body fluids.People will be exposed to DEHP.DEHP can be rapidly transformed into the main toxic metabolite Mono-2-ethylhexyl ester(MEHP)in gastrointestinal tract by esterase after oral administration.It can be distributed in the liver,kidney,pancreas and reproductive organs.Finally,it is harmful to human health.The absorption and distribution of exogenous compounds in target organs can determine their toxicity.The first contact of PAEs is through oral intake.Therefore,it is of great value to study the absorption of PAEs in the intestine for guiding the dosage of PAEs.The factors affecting intestinal absorption include the physiological and pathological state of the intestine itself and the physicochemical properties of exogenous compounds.Since the physical and chemical characteristics of a specific PAEs are certain,the internal exposure dose depends on the physiological and pathological state of the intestinal tract after determining the external exposure dose.The physiological and pathological state of intestinal tract is closely related to intestinal barrier.Intestinal barrier includes four parts: biological barrier,intestinal epithelial barrier,mucus barrier and immune barrier.which is the first gateway for exogenous substances in the digestive tract to enter the human body.Each barrier has a different structure and biological function,they maintain the body from the infection of exogenous substances.It is the first gate for exogenous substances entering the digestive tract to enter the human body.Therefore,food additives,such as dietary emulsifiers,which affect the pathophysiological state or intestinal microecological homeostasis,have become a research hotspot.Food emulsifier is widely used in food industry.It can enhance the consistency of food and improve the taste of food.Synthetic dietary emulsifier polysorbate 80(P80)is a common non-ionic food emulsifier,which is suitable for the most common food,such as ice cream,cake,milk powder and cheese.Emulsifier P80 can change the composition and distribution of gut microbiota,and lead to the absorption of many toxic and harmful substances including emulsifier.However,current studies have not provided sufficient evidence to elucidate the mechanism of emulsifier promoting plasticizer absorption and the relationship between low-dose food emulsifiers,environmental endocrine disrupting chemicals(EEDs)and intestinal barrier.AimResearch the emulsifier P80 to promote the absorption and distribution of MEHP,and further explore the mechanism of P80 to promote the absorption of MEHP in vivo and in vitro,revealing the relationship between the emulsifier P80,the plasticizer MEHP and the intestinal mucosal barrier.Methods1.Methodology validation of MEHP-AF microplate fluorescence detection methodAccording to the synthesis method of MEHP-AF designed by our group,we crosslinked Mono-2-ethylhexyl phthalate(MEHP)with 5-aminofluorescein(5-AF)in the presence of HOBt/EDCI,and synthesized MEHP-AF,which is a fluorescent probe for in-situ tracing of MEHP.Plasma solutions with different final concentrations of MEHPAF were prepared.The OD value was detected by microplate reader.The standard curve was drawn,and the linearity and detection limit were calculated.The OD values of samples were detected at different time points to evaluate the stability of samples.The accuracy,precision,sensitivity and robustness to interference were calculated by repeated operation of the same person and operation of different persons.2.The influence of P80 on the absorption and distribution of MEHP-AFSD rats were divided into two groups(n=3)and single gavage with 5 mg/kg MEHP-AF or 25 mg/kg P80 + 5 mg/kg MEHP-AF.The peripheral blood of orbital venous plexus was collected at 15,30,45,60,90,120,180,240,360,480,600,720,1440 min respectively.The OD value of plasma was measured and the concentration was calculated according to the standard curve of MEHP-AF.Phoenix 8.1 was used to calculate the toxicokinetics parameters by non-compartmental method and compartmental method.And the bioavailability was compared.Another six week old ICR male mice were divided into nine groups((1)blank group,(2)corn oil group,(3)5mg/kg P80 group,(4)15 mg/kg P80 group,(5)25 mg/kg P80 group,(6)MEHP-AF group,(7)5 mg/kg P80 + 3 mg/kg MEHP-AF group,(8)15 mg/kg P80 + 3 mg/kg MEHP-AF group,and(9)25 mg/kg P80 + 3 mg/kg MEHP-AF group),the corresponding doses of P80 and MEHP-AF were given by gavage every day for 2 weeks.The weight changes of mice were detected every day.90 minutes after the last gavage,the orbital venous plexus blood samples of group(6)-(9)were taken to measure the plasma concentration of MEHP-AF.The heart,liver,spleen,lung,kidney,testis and other organs were weighed and histologically observed.The small intestine tissues of mice were taken and frozen sections were prepared.The intestinal uptake of MEHP-AF was observed under laser confocal scanning microscope.Human colorectal adenocarcinoma cells(Caco-2)and human colon cancer cells(HT-29)were selected as the model cells for this study.Different doses of P80(0.25,0.5,1.0,2.0,3.0 mg/mL)were used to treat the two kinds of cells respectively for 24 hours,the two kinds of cells were incubated with 1 μM MEHP-AF for 1 hour,and the uptake of MEHP-AF was observed by laser confocal microscopy.3.The effect of P80 on intestinal mucus barrierThe small intestine samples of group(1)-(5)were prepared for paraffin section,and the formation of viscous substances was detected by AB-PAS staining.In addition,small intestine tissues of each group were taken,frozen sections were prepared,and the expression of Muc-2 protein was observed by immunofluorescence staining.HT-29 cells were cultured with different concentrations of P80(0.25,0.5,1.0,2.0,3.0 mg/mL)for 24 hours.The mucin(Muc-2)secreted by HT-29 cells was quantitatively analyzed by human Muc-2 ELISA kit.4.The effect of P80 on intestinal epithelial barrierSmall intestine tissue immunohistochemical staining and Caco-2 cell immunofluorescence staining were used to observe the formation of tight junction proteins claudin-1 and occludin.Western blot and qPCR were used to detect the expression of claudin-1 and occludin at protein and mRNA levels.5.The effect of P80 on intestinal permeabilityIn order to detect the intestinal permeability of mice,after oral administration of FITC-dextran(600 mg/kg)for 4 h,the blood samples of orbital meridian plexus were taken to measure the OD value of plasma to obtain the concentration of FITC-dextran in plasma.Caco-2 monolayer model was used in vitro.The monolayer was treated with different concentrations of P80(0.25,0.5,1.0,2.0,3.0 mg/mL)for 48 h.The Transepithelial electrical resistance(TEER)values was measured.FITC-dextran was added to the upper layer of Transwell and incubated for 2 h.The OD value of the lower layer was detected to obtain the concentration of FITC-dextran in the lower layer.Results1.Methodology validation of MEHP-AF microplate fluorescence detection methodMEHP-AF was successfully synthesized by amidation of MEHP and 5-AF in HOBt/EDCI system.The methodological validation showed that there was no endogenous interference in the quantitative analysis of MEHP-AF in blood,and the established MEHP-AF fluorescence detection method could be used for the concentration analysis of MEHP-AF in plasma.2.The influence of P80 on the absorption and distribution of MEHP-AFThe results of toxicoy experiment showed that the concentration of MEHP-AF in plasma of P80 + MEHP-AF group was significantly higher than that in MEHP-AF group,P80 could significantly promote the absorption of MEHP-AF and improve its bioavailability.After two weeks of continuous administration of P80,the concentration of MEHP-AF in plasma was also significantly increased.There was no significant difference in weight between the mice in each group,and there was no obvious damage to the organs of heart,liver,spleen,lung and kidney.The results of frozen sections of small intestine showed that MEHP-AF uptake was more obvious in P80 group.P80 significantly promoted the uptake of MEHP-AF in both Caco-2 cells and HT-29 cells.3.The effect of P80 on intestinal mucus barrierThe results of AB-PAS staining showed that P80 changed the composition and structure of intestinal mucilage.Immunofluorescence staining of Muc-2 showed that P80 inhibited the expression of mucin-2,and the higher the concentration of P80,the lower the expression of Muc-2.The same results were obtained by ELISA.The amount of Muc-2 protein secreted by HT-29 cells treated with P80 was significantly decreased.4.The effect of P80 on intestinal epithelial barrierImmunohistochemical staining showed that P80 decreased the expression of tight junction protein in intestinal epithelial cells.The higher the concentration of P80,the lower the expression of tight junction protein.Similarly,immunofluorescence staining at the cellular level yielded similar results.Western blot and qPCR results showed that P80 decreased the expression of tight junction associated protein and mRNA in small intestine and Caco-2 cells at both protein and mRNA levels.5.The effect of P80 on intestinal permeabilityFTTC-dextran flux and TEER value showed that P80 could reduce TEER value and increase FTTC-dextran flux of Caco-2 cell monolayer,that is,increase intestinal permeability,and the higher the concentration of P80,the greater the intestinal permeability.ConclusionDietary emulsifier P80 can destroy the intestinal mucosal barrier,reduce the secretion of mucus,destroy the tight junction of intestinal epithelial cells,and ultimately increase the intestinal permeability,thus promoting the intestinal absorption of plasticizer MEHP.