Study On The Mechanism And Application Of The Excitatory Effect Of Glucosamine On Intestinal P-glycoprotein

P-glycoprotein(P-gp),also known as ABCB1 protein,is a transmembrane glycoprotein with a molecular weight of approximately 170 k Da,encoded by the multidrug resistance 1(MDR1)gene.As the first representative member of the ATPbinding cassette(ABC)transporter superfamily discovered in humans,P-gp is widely expressed on the surface of epithelial cells in human intestine and blood-brain barrier.Pgp is a powerful drug efflux pump with multiple cooperative drug binding sites.It relies on ATP energy supplyment and can non-specifically pump a large number of drug molecules out of cells quickly,thereby protecting cells from harmful molecules from the external environment.In addition,P-gp can efflux various anticancer drugs and hydrophobic drugs,resulting in a decrease in intracellular drug concentration.In particular,overexpression of P-gp in tumor cells can seriously affect drug efficacy,leading to multidrug resistance.Hence,P-gp inhibition is a crucial target and can be used to develop anticancer therapy.However,it is worth noting that some studies have shown that activating this efflux defense mechanism,especially intestinal P-gp activity,in which it can effectively prevent the absorption of harmful xenobiotics and is of great significance for detoxification and organ protection.There are few studies on P-gp activators in the existing literature,and commonly used substances with activating functions on P-gp,such as dexamethasone and verapamil,produce serious toxic reactions themselves when administered in large amounts or long time,thereby limiting their application scope.Therefore,developing safe and effective new P-gp activators is of great value for detoxification research.In the preliminary research of our research group,it was found that glucosamine can rapidly activate the efflux activity of P-gp,improve its transport efficiency for foreign drug molecules,and thus reduce the oral bioavailability of drugs.Therefore,confirming glucosamine as an activator of P-gp,exploring its activation mechanism,and exploring its possible application as a P-gp activator are of great significance.Glucosamine(GlcN)is the most abundant naturally occurring amino sugar,formed by the substitution of an amino group for a hydroxyl group of glucose.It is widely present in animal exoskeletons and bacterial cell walls as a derivative and is soluble in water and hydrophilic solvents.GlcN is an essential substance for the synthesis of protein polysaccharides in human joint cartilage matrix.Various studies have reported its multiple physiological activities,including it is used for the prevention and treatment of osteoarthritis throughout the body,it plays an important role in protein glycosylation processes,and it acts as a major component of some glycoconjugates.P-gp is a representative cell membrane glycoprotein.Therefore,exploring the interaction between GlcN and P-gp and elucidating its activation mechanism for P-gp efflux transport can guide the further rational application of GlcN and provide a theoretical basis for the development of it as the detoxifying agent.The main research content of this dissertation is divided into the following three parts:Part I: The discovery of the excitatory effect of glucosamine on P-glycoprotein.Through rat pharmacokinetics,it was found that GlcN showed a significant inhibitory effect on the oral absorption of capecitabine,reducing its bioavailability by 60.40%.In addition,the influence of GlcN on the stability of capecitabine was excluded by determining the content change.Subsequently,the effect of concentration and action time on the inhibition of capecitabine intestinal absorption by GlcN was investigated using the everted intestinal sac method.A 20-minute treatment with 50 mg/m L GlcN resulted in the lowest absorption of capecitabine in the isolated small intestine.The effects of GlcN were also evaluated on the oral bioavailability of acyclovir,cimetidine,and oxcarbazepine under these conditions.The GlcN inhibited the oral absorption of all three drugs,reducing their bioavailability by 63.37%,64.92% and 76.40%,respectively,suggesting that GlcN inhibits non-specifically the intestinal absorption of drugs.Then,the cytotoxicity of GlcN on human colorectal cancer cell line Caco-2 was examined by CCK-8 method.Incubation of Caco-2 cells with GlcN below 20 m M concentration for 24 hours and 48 hours did not show any significant cytotoxicity.In order to investigate the transport of capecitabine on monolayers of Caco-2 cells,a Transwell model was created.Capecitabine trans-cellular transport was inhibited by GlcN,but promoted by verapamil,an inhibitor of Pglycoprotein.The results exhibited that the inhibition effect of GlcN might be related to the efflux and transport of P-gp.Part II: Experimental confirmation and mechanism of glucosamine on Pglycoprotein activation.The effects of GlcN on the intracellular accumulation and outflow of Rhodamine 123(Rh123),a fluorescent substrate of P-gp,were determined by enzyme markers.The results showed that GlcN could inhibit the intracellular accumulation of Rh123 by enhancing the outflow of Rh123,and the inhibition of GlcN accumulation disappeared after silencing the expression of P-gp by si RNA,confirming that its inhibition of intracellular accumulation of Rh123 was linked to P-gp.The recombinant human everted vesicle method proved that GlcN can promote the transport efficiency of P-gp to substrate.Furthermore,the interaction between GlcN and P-gp was confirmed by the cell thermal shift experiment.Through molecular docking,we explored the possibility of binding GlcN and its polymeric derivatives to P-gp,which was then confirmed by surface plasma resonances(SPRs)to investigate the structural effects of GlcN.Considering GlcN’s overall effect on intestinal biochemical barrier,its effects on CYP3A4 and P-gp-mediated ATPase activity were investigated.It was investigated whether the activation effect of GlcN on intestinal P-gp is the direct target of its inhibition of drug absorption at the biological level,by comparing the effects of GlcN on digoxin,the classical substrate of P-gp,in SD rats and gene knockout mice.It was found that GlcN can significantly reduce the bioavailability of digoxin in SD rats,but not inhibit the absorption of digoxin in P-gp gene knockout mice.This experiment showed that GlcN can directly bind to P-gp,which may change P-gp conformation and enhance its other binding sites on substrate transport.Thus,GlcN can rapidly activate intestinal P-gp in a short time,enhance its efflux transport activity,and inhibit the intestinal absorption of drugs.Therefore,it is expected that GlcN can be used as a safe and effective P-gp activator for detoxification research.Part III: The application of glucosamine(GlcN)on the activation of P-glycoprotein(P-gp).The aforementioned study on GlcN’s quick and efficient activation of intestinal Pgp serves as a reference for justifying the use of GlcN as a P-gp activator in detoxification research.First off,it is evident that GlcN is widely present in the chitosan oligosaccharide products currently on the market after analyzing the level of polymerization of various brands,and the impact of these different levels of polymerization on the pharmacokinetics and pharmacokinetics of capecitabine demonstrates that the presence of GlcN and oligosaccharides in the mixture should be reasonably avoided when using them.The rat model of paraquat poisoning was developed,and GlcN’s effects on the pharmacokinetics and survival rate of rats were examined.It was discovered that paraquat could significantly lower the blood concentration of paraquat in the model rats,extend their survival cycle,and increase their survival rate,indicating that GlcN could be developed as a safe and effective antidote as a P-glycoprotein activator.In conclusion,this study demonstrates that GlcN can inhibit oral drug absorption by triggering the intestinal P-gp efflux effect and investigates the mechanism of GlcN activating P-gp.On the other hand,this study reveals the activation effect and mechanism of GlcN on P-gp,which facilitates rationalizing GlcN use scenarios.Furthermore,it provides an opportunity to conduct detoxification research using P-gp activation effects.