Study On The Mechanism Of Oral DhHP-6 Ameliorating Hepatic Insulin Resistance In Type 2 Diabetes Mellitus

Type 2 diabetes mellitus(T2DM)is a common multifactorial metabolic disease,characterized by hyperglycemia and insulin resistance.T2 DM has increased in prevalence and emerged as one of the largest public health issues globally.Oxidative stress is due to the overproduction of reactive oxygen species(ROS),and excess ROS leads to cellular dysfunction,encompassing impaired energy metabolism,altered cell signaling,impaired cell transport mechanisms and inflammation.Chronic oxidative stress is the underlying factor that leads to the development of insulin resistance,dysregulated pathways of metabolism and diabetes.Thus,the development of anti-oxidative stress drugs may serve as a new option in treating type 2 diabetes.Deuterohemin-?Ala-His-Lys(Dh HP-3)is a novel peroxidase mimic that was designed to be based on microperoxidase-11 in our laboratory.Previous studies have found that Dh HP-3 can decrease blood glucose in type 2 diabetic rats and inhibit high glucose-induced rat pancreatic INS-1 ? cell apoptosis by activating the PI3K/AKT signaling pathway.However,due to its short half-life and rapid plasma clearance,Dh HP-3 was administered via injection,which may result in poor patient compliance and risk of infection.Hence,oral delivery of polypeptide drugs is still the most ideal route of administration.In this study,Deuterohemin-?Ala-His-Thr-Val-Glu-Lys(Dh HP-6)was designed and synthesized.Previous studies have indicated that the relative enzyme activity of Dh HP-6 was higher than that of Dh HP-3.Additionally,Dh HP-6 had strong stability in artificial gastrointestinal fluid and plasma,in which the relative enzymatic activity of the degradation products was similar to Dh HP-6.In order to explore the effect of oral DHHP-6 on type 2 diabetes,this paper was mainly conducted according to three aspects.In the present study,a T2 DM mice model was established by introducing a high-fat diet combined with streptozotocin to investigate the effect of oral Dh HP-6 on T2 DM.The results demonstrated that oral Dh HP-6 could effectively reduce blood glucose and improve insulin resistance in T2 DM mice.Moreover,serological studies have found that Dh HP-6 can increase the activity of SOD GSH and CAT and reduce MDA content,thereby improving oxidative stress.In addition,Dh HP-6 was also found to improve dyslipidemia in T2 DM mice by reducing T-CHO,TG and LDL-C while increasing HDL-C.Liver function indexes(AST,ALT and ALP)and liver morphology showed that Dh HP-6 could effectively maintain the structural and functional integrity of livers in T2 DM mice.Furthermore,by observing islet and kidney morphology,Dh HP-6 was found to effectively improve islet atrophy and vacuolar degeneration while maintaining the integrity of the glomerular structure in T2 DM mice.Notably,Dh HP-6 was observed to significantly reduce FBPase and G6 Pase and increase HK in T2 DM mice.Liver PAS staining showed that Dh HP-6 effectively promoted glycogen synthesis in T2 DM mice.Additionally,Western Blot analysis of liver proteins showed that Dh HP-6 promoted phosphorylation of IRS-1 at Tyr632,AKT at Ser473 and GSK-3? at Ser9 and inhibited the phosphorylation of IRS-1 at Ser307,thus activating the PI3K/AKT signaling pathway in the livers of T2 DM mice.At the same time,Dh HP-6 promoted the phosphorylation of AMPK at Thr172 and inhibited the expression of G6 Pase and PEPCK while activating the AMPK signaling pathway in T2 DM mice.In addition,Dh HP-6 was found to promote the expression of Nrf2 and inhibit the expression of Keap1,improving the antioxidant systems of T2 DM mice.Therefore,Dh HP-6 can activate the PI3K/AKT,AMPK and Nrf2-Keap1 signaling and improve insulin resistance symptoms in T2 DM mice.In order to further explore the mechanism of Dh HP-6 in improving hepatic insulin resistance,a Hep G2 cell insulin resistance model was established and induced by glucosamine.The results demonstrated that Dh HP-6 could effectively improve cell damage and promote glucose absorption and glycogen synthesis in the Hep G2 cell insulin resistance model.Dh HP-6 was also observed to inhibit ROS production and improve mitochondrial dysfunction in the Hep G2 cell insulin resistance model.Western Blot analysis showed that Dh HP-6 could activate the PI3K/AKT,AMPK and Nrf2-Keap1 signaling pathways in the Hep G2 cell insulin resistance model.After LY294002,a PI3 K specific inhibitor,was added,PI3K/AKT signaling was not completely inhibited,and LY294002 partially inhibited AMPK and Nrf2-Keap1 signaling.When the AMPK inhibitor compound c was added,AMPK signaling was also not completely inhibited,and PI3K/AKT and Nrf2-keap1 signaling pathways were partially inhibited.Therefore,PI3K/AKT,AMPK and Nrf2-Keap1 signaling did not act independently,rather,they promoted each other.Thus,Dh HP-6 can activate the PI3K/AKT,AMPK and Nrf2-Keap1 signaling to improve insulin resistance in glucosamine-induced Hep G2 cells.In order to further explore the oral delivery strategy of Dh HP-6,folate-carboxymethyl chitosan(FA-CMC)targeting folic acid receptors were synthesized,and the microsphere composed of FA-CMC,cell-penetrating peptide(CPP)and Dh HP-6 was prepared.Additionally,the feasibility of the microsphere as an oral delivery system for Dh HP-6 was verified.Compared to CPP(FI-6 and LK-18),RR-9 was found to significantly reduce the transmembrane resistance of the Caco-2 cell model at the same concentration.Therefore,RR-9 was selected for the subsequent experiments.Folate-carboxymethyl chitosan exhibited almost no toxicity to Caco-2 cells.Moreover,the adhesion ability to Caco-2 cells was stronger than that of carboxymethyl chitosan.Dh HP-6-CPP-folate-carboxymethyl chitosan microspheres was released slowly in simulated intestinal fluid,which then became significantly faster in simulated gastric fluid.Dh HP-6-CPP-folate-carboxymethyl chitosan microspheres was also found to significantly promote the transmembrane transport of Dh HP-6 in the Caco-2 cell model.Therefore,Dh HP-6-CPP-folate-carboxymethyl chitosan microspheres were observed to enhance the adhesion ability of chitosan and promote the transmembrane transport of Dh HP-6 in the Caco-2 cell model.In summary,Dh HP-6 could ameliorate hepatic insulin resistance in type 2 diabetes mellitus by activating PI3K/AKT,AMPK and Nrf2-Keap1 signaling.Furthermore,adopting Dh HP-6-CPP-folate-carboxymethyl chitosan microspheres as a Dh HP-6 oral delivery system was determined to be feasible.