Study On The Mechanism Of ACE Inhibitory Peptide By Ferritin-Alginate And Cell Absorption

In this paper,horse spleen ferritin(HSF)and alginic acid(SA)were used as nano-embedded carriers for encapsulation of ACE inhibitory peptide alanine-histidine-leucine-leucine(Ala-His-Leu-Leu,AHLL),using the reversible assembly properties of ferritin under extremely acidic conditions and the controlled release of sodium alginate,expecting to improve the absorption of the ACE-inhibiting peptide AHLL in the digestive system.The encapsulation efficiency is used as an evaluation index to optimize the test conditions.Nanoparticle size and transmission electron microscopy were used to observe the characterization of nanoparticles.The spectral changes were used to compare the changes of protein structure after embedding treatment,and the effects of UV radiation,temperature and pH on the stability of nanoparticles were studied.After simulating the absorption of AHLL nanoparticles by in vitro digestion,a Caco-2 cell monolayer model was established and validated,and the model was used to study the intestinal absorption mechanism of HSF-AHLL.The main conclusions of this paper are as follows:(1)The optimal conditions for preparing nanoparticles are:carrier ferritin concentration between 1-2?M,polysaccharide concentration 10 mg/L,and final concentration of AHLL in the range of 100-200?g/mL,under which the nanoparticle system was more stable.Through particle size and potential measurement,AHLL is likely to be embedded in the HSF cavity and characterized by transmission scanning electron microscopy.It is apparent that HSF-AHLL and HSF-SA-AHLL form a stable nanosystem.(2)UV spectroscopy indicates that AHLL nanoparticles have a macromolecular conjugated structure similar to HSF.Fluorescence spectroscopy indicates that AHLL nanoparticles only change the tryptophan microenvironment of ferritin on the quadruplex channel.It can be seen from the circular dichroism that the content of?-helical structure and?-turn structure of the protein is significantly reduced after embedding treatment,and the content of?-sheet structure is increased,indicating that the spatial structure of the protein after embedding changes to some extent,thus the protein is more developed.After UV irradiation for 24 h,the degradation rate of HSF-AHLL and HSF-SA-AHLL was 35.83%and 43.86%lower than that of AHLL alone.The degradation rate at 80°C decreased by26.96%and 38.51%respectively after 60 min treatment at different temperatures,indicating that HSF-AHLL and HSF-SA-AHLL are significantly improved in light and thermal stability compared to free AHLL.The effect of pH on the stability of nanoparticles indicates that the stability of HSF-AHLL nanoparticles is best in neutral environment.The DSC experiment shows that the ferritin structure after embedding treatment is more developed,and the interaction with the AHLL hydrogen bond is strengthened,and the thermal stability of the nanoparticles is better.In vitro simulated gastrointestinal system found that HSF-AHLL enters the digestive system compared to AHLL alone in the extremely acidic environment of the gastric tract,and does not improve ACE inhibitory activity as a whole;After HSF-SA-AHLL entering the gastric tract,the sustained release protection of SA makes AHLL not digested by pepsin,and HSF-AHLL enters the intestine smoothly,and the stability is better under the neutral condition of the intestinal tract,so that it is effectively absorbed,and the ACE inhibitory activity is remarkable.(3)The results of Caco-2 cell model established in our laboratory are as follows:Caco-2 cells have a growth cycle of about 7 to 9 days,growth is slow,the passage time is controlled between 4 and 5 days,and the digestion time is controlled at 3 to 5 minutes.Between 4 and5 days,Caco-2 cells were observed to form a dense monolayer structure by an inverted microscope.After 16 days of culture,Caco-2 cells differentiated into a monolayer model similar in morphology to small intestinal epithelial cells,forming a dense and dense structure between the microvilli and the cells.The transmembrane resistance of the single-layer model is basically maintained at around 650?·cm~2.The alkaline phosphatase activity of the monolayer side of Caco-2 cells was 17.71 times of the alkaline phosphatase activity on the basal side,indicating the obvious polarization phenomenon.The transporting experiment of the fluorescent yellow AP?BL side also has a permeability coefficient lower than 1.0×10-6 cm/s as specified in the transport experiment.(4)Using the validated Caco-2 cell model to study the cellular intestinal absorption mechanism of HSF-AHLL nanoparticles:Cell transport experiments were performed by HST-AHLL at a concentration of 100-200?g/mL by MTT assay.At the same time,the HSF-AHLL nanoparticles were significantly more transported on the Caco-2 cell monolayer model than the AHLL alone,and the apparent permeability coefficient(120 min)was also significantly higher than the latter.The two-way transport of HSF-AHLL is obviously dependent on time,and active transport and passive transport exist simultaneously.The apparent permeability coefficient of HSF-AHLL transported in the AP?BL direction is26.97×10-6 cm/s,while the BL?AP direction is 5.79×10-6 cm/s.The transport rate from the AP side to the BL side is much larger than the reverse direction.The transport rate indicates that the absorption of HSF-AHLL in Caco-2 cell monolayer absorption is greater than that of efflux and has a certain directionality.The transport of HSF-AHLL in the Caco-2 cell model is related to both temperature and pH and is dependent on sample concentration.The final concentration of AHLL in HSF-AHLL nanoparticles was 100?g/mL,the pH of the feed was 7.4,and the transport permeability coefficient was the highest at 37°C.The results of the addition of the efflux protein inhibitor indicated that AHLL is an efflux substrate for P-glycoprotein and its efflux is mainly mediated by the MRP2 transporter.Endocytosis inhibitors and peptide carrier inhibitors had no significant effect on the transport of AHLL,whereas sodium deoxycholate significantly promoted its transport,demonstrating that AHLL is primarily transported across the intestinal epithelial cells via cell bypass.These two inhibitors and sodium deoxycholate did not significantly promote the transport of HSF-AHLL,possibly by other carriers.