The Study Of The Complex Of Lactoferrin And Lentinus Edodes Polysaccharide Inhibiting Pancreatic Island β Cell Damage And The Molecular Mechanisms

One of the main pathogenesis in type II diabetes is pancreaticβcell damage.The usage of natural active molecules is an effective way to protect pancreatic islandβcells.Bovine lactoferrin（BLF）has many biological activities including anti-inflammatory,anti-oxidation and hypoglycemic.The BLF has a potential protective effect on pancreatic islandβcells.However,it is sensitive to changes in physical and chemical properties in the physiological environment and has low bioavailability in vivo.Lentinus edodes mycelia polysaccharide（LMP）is an active polysaccharide with antioxidant activity that identified in our laboratory.It can be used to maintain the stability of protein molecules.In this study,bovine lactoferrin and Lentinus edodes mycelia polysaccharide were used to prepare BLF-LMP complex.The interaction mechanism of the BLF-LMP complex was explored by multi-spectroscopy.By detecting the inhibitory effect and mechanism of the complex on hydrogen peroxide（H₂O₂）glucose-induced cell injury,it provides a theoretical basis for its application in inhibiting pancreaticβcell injury.In order to study the formation mechanism of the BLF-LMP complex,the quenching mechanism of BLF by LMP was investigated by fluorescence spectroscopy.The type of interaction force of the complex was calculated according to thermodynamic results.Ultraviolet spectroscopy（UV-Vis）,three-dimensional fluorescence spectroscopy（3D spectra）,synchronous fluorescence spectroscopy,Fourier transform infrared spectroscopy（FT-IR）and circular dichroism（CD spectra）were used to explore the interaction mechanism between LMP and BLF.Then,in vitro digestion simulation experiments were used to explore whether LMP inhibited the degradation of BLF in the gastric environment.In order to explore the protective effect of the BLF-LMP complex on pancreatic islandβcells,mouse pancreaticβcell line MIN6 cells were selected as the research object.The appropriate concentration of H₂O₂ and glucose injury was selected by MTT method,and the effect of the complex on H₂O₂/glucose-induced cell injury was detected.The level of reactive oxygen species（ROS）was used to detect the effect of BLF-LMP complex on the oxidative stress level of cells exposed to H₂O₂/glucose.Hoechst33258 staining assay was used to detect the effect of the complex on apoptosis.The effect of the BLF-LMP complex on H₂O₂/glucose-induced was detected by Western Blot.The results showed that lentinan could quench the endogenous fluorescence of lactoferrin（static quenching）,and the two were spontaneously combined by electrostatic force.Synchronous fluorescence spectroscopy and 3D spectroscopy showed that the addition of LMP changed the microenvironment of BLF amino acid residues.The results of FT-IR showed that the secondary structure of protein changed after the combination of LMP and BLF.The results of in vitro digestion simulation experiments showed that the addition of LMP effectively inhibited the degradation of BLF in the gastric physiological environment.Biological experiments showed that a certain concentration of H₂O₂（400μM）and glucose（40m M）could reduce cell viability and induce apoptosis.The BLF-LMP complex（0.0125,0.025 and 0.05 m M）can effectively improve the cell viability,and there is a dose effect between them.Compared with the monomer,the BLF-LMP complex significantly inhibited apoptosis.Under the action of H₂O₂/glucose,the intracellular ROS level increased and the intracellular oxidative stress level increased.With the addition of the BLF-LMP complex,ROS content decreased,indicating that the complex can effectively inhibit H₂O₂/glucose-induced oxidative stress.Western Blot results showed that the complex could significantly inhibit the up-regulation of Bax/Bcl-2 ratio induced by H₂O₂/glucose,the activation of MAPK and NLRP3signaling pathways,and the nuclear transfer of NF-κB.The above results show that BLF-LMP complex can effectively inhibit MIN6 cell apoptosis and pyroptosis caused by oxidative stress and inflammation.In summary,the LMP can form a complex with BLF and inhibit the digestion of BLF.The BLF-LMP complex alleviates H₂O₂/glucose-induced apoptosis and pyroptosis by regulating oxidative stress and inflammatory signaling pathways.This study elucidated the interaction mechanism of the BLF-LMP complex and the activity of the complex to inhibit oxidative stress and inflammation in pancreatic islandβcells.The study provides a fresh perspective for the application of the protein-polysaccharide complex in inhibiting pancreaticβcell injury.