Screening,Effect And Mechanism Of Key Enzyme Inhibitiors For Neu5Gc Synthesis

N-glycolylneuraminic acid(Neu5Gc)is a non-human sialic acid that is ubiquitous in many mammals other than humans,such as horses,dog serum,and gorillas.However,human consumption of foods rich in Neu5Gc,such as red meat,will produce heterologous autoantigens and accumulate in body,which in turn stimulates the body to cause inflammation and accelerate the onset of tumors and related diseases.Therefore,if a suitable pretreatment method is found before slaughtering,especially in the stagnation,to reduce the content of red meat,it will have a profound impact on the safe production of the red meat supply chain.Therefore,this paper intends to screen the enzyme inhibitors that effectively reduce the synthesis of Neu5Gc from the pre-slaughter treatment of animals,and to study its effects and mechanisms.(1)In vitro screening of synthetic key enzyme inhibitorsThrough literature review,it was determined that luteolin,kaempferol and quercetin were used as experimental reagents to test the endocytic inhibition of rat primary submandibular gland epithelial cells(RAT-iCELL-g009)and prostate cancer cells(PC3)showed that luteolin and kaempferol Quercetin can inhibit the release of free Neu5Gc into cells by cell pinocytosis or endocytosis.Compared with the control group,the content of Neu5Gc in the primary submandibular gland epithelial cells(RAT-iCELL-g009)in the experimental group decreased by 35.77±2.75%,37.59±2.52% and 29.77±2.9%,respectively.The content of Neu5Gc in the prostate cancer cells(PC3)cells in the experimental group decreased by 34.98±2.74%,42.25±1.45% and 42.21±0.66% respectively.(2)Study on inhibition of Neu5Gc synthesis in ratsOn the basis of previous experiments,the intervention effect of Neu5Gc synthesis in rats in vivo was explored by using luteolin 150 mg/(kg·d),kaempferol 200 mg/(kg·d),quercetin 200 mg/(kg· d)to feed rats for 60 days.Then the content of Neu5Gc in the muscle tissue,liver and kidney of rats after gavage for 30 and 60 days was detected by HPLC.The results showed that: At 30 days,compared with the control group,luteolin,kaempferol and quercetin inhibited the synthesis of Neu5Gc in muscle tissue significantly.The maximum inhibition rate was 13.36±0.09%(P<0.05).The inhibition of Neu5Gc synthesis in kidney and liver was not significant.On the 60 th day,compared with the control group,the inhibitory effect of luteolin,kaempferol and quercetin on the synthesis of Neu5Gc in muscle tissue was weakened,and the inhibitory effect on the content of Neu5Gc in the kidney and liver was enhanced.The maximum inhibition rate was 34.35±0.09%(P<0.05);Therefore,the optimal rate of gastric perfusion was determined to be 30 days after calculation of inhibition rate and comprehensive evaluation of daily diet.(3)Relationship between Neu5Gc synthesis and inhibitor concentration in ratsThe intragastric cycle was chosen to be 30 days,and the optimal inhibitor concentration was determined by single factor experiment.Compared with the control group,the optimal concentration of luteolin inhibiting the synthesis of Neu5Gc in muscle tissue and liver of rats was 150 mg/(kg·d),and the inhibition rates were 13.48±1.83% and 12.8±0.06%,respectively.The inhibition of Neu5Gc synthesis in rat kidneys increases with increasing inhibitor concentrations.The concentration of kaempferol has a certain inhibitory effect on the synthesis of Neu5Gc in rats,and there is a dose-effect relationship between the inhibition of Neu5Gc in muscle tissue.The optimal concentration of quercetin to inhibit Neu5Gc synthesis in rat muscle tissue,liver and kidney was 200 mg/(kg·d),and the inhibition rates were 24.74±0.07%,14.79±0.09%,and 5.82±0.02%,respectively.(4)Molecular simulation of the mechanism of sialyltransferase inhibiting the synthesis of Neu5GcMolecular docking and molecular dynamics simulation methods were used to explore related inhibition mechanisms.The molecular docking two-dimensional interaction diagram showed that luteolin,kaempferol and quercetin effectively occupied the active site of the 5' cytidine monophosphate specific inhibitor of sialyltransferase.The 200 ns kinetic simulation of the complex indicated that the residues of hydrogen bonding with sialic acid transferase and luteolin were mainly: Glu305,Asn150,and the residues of hydrogen bonding with pentachlorophenol were mainly His301 and Gly298 The main residues of hydrogen bonding with quercetin are Gly293,Glu324,Thr272 and Ser276.The combination of free energy decomposition indicates that van der Waals forces,electrostatic interactions,and hydrogen bonding play an important role in the inhibition process.