Effects Of Advanced Glycation End Products On Autophagy Of BMSCs And Its Mechanism Initially Explored

Background: Diabetes is a clinically common metabolic disease.In recent years,the incidence of diabetes has also risen significantly,becoming one of the major chronic diseases in China.And the age of patients is showing a trend of getting younger and younger.The continuous increase of blood glucose in patients with diabetes can cause chronic damage and dysfunction of various tissues and organs.When it reaches the bone tissue,it often causes osteoporosis.AGEs refer to stable molecules produced by proteins and other macromolecules spontaneously reacting with glucose or other reducing monosaccharides through non-enzymatic glycosylation without the participation of enzyme Covalent adducts,long-term poor glycemic control in patients with diabetes can lead to excessive generation of AGEs.There are two sources of AGEs in the body,one is that protein and excess sugar are combined in the body,and the other is that AGEs present in food are taken into the body.Because AGEs are specific and stable to enzymes,they are not easily removed,and they gradually accumulate in the body as they age.Studies have shown that with the excessive accumulation of AGEs in the bone,it will affect the normal function of osteoblasts and is one of the important factors leading to diabetic osteoporosis.The study of advanced glycation end products may provide a new direction for the treatment of osteoporosis in patients with diabetes.And how does AGEs affect the function of osteoblasts? What is its mechanism of action? AGES mainly causes damage to the body through three mechanisms: 1.Destructing its structure and function through direct cross-linking and binding with macromolecular substances such as proteins,lipids,nucleic acids,etc.2.Changing the effect on signal transduction pathways and enzyme activity 3.Interacts with cell surface AGEs receptor RAGE,triggering biological effects.autophagy is a process in which a cell wraps some of its own proteins and organelles in a specific membrane structure,sends it to the lysosome for degradation,and makes it available for reuse by the cell.AGEs can directly cross-link with proteins in the body to cause changes in autophagy levels of BMSCs cells,it can also bind to its receptor RAGE to activate a series of signaling pathways in BMSCs cells,thereby triggering changes in cell autophagy activity and affecting BMSCs.Osteogenic differentiation leads to osteoporosis.Autophagy is also negatively regulated by the PI3 K / Akt / m TOR pathway.In the case of adequate cell nutrition,autophagy is inhibited by inhibiting the autophagy-related gene ATG1.AGEs can activate the PIK3 / AKT / m TOR signaling pathway and phosphorylate AKT,thereby inhibiting the autophagy response of BMSCs.The endoplasmic reticulum is an important organelle for protein synthesis,folding,and secretion in eukaryotic cells.The stability of the environment within the endoplasmic reticulum is the basic condition for realizing the function of the endoplasmic reticulum.Therefore,the endoplasmic reticulum has a very strong homeostasis system.However,there are still many factors that can lead to an imbalance in the homeostasis of the endoplasmic reticulum function,resulting in endoplasmic reticulum stress(ERS).The literature indicates that the accumulation of AGEs in humans may also cause cell damage,inflammation and apoptosis by inducing endoplasmic reticulum stress,and the degree of ERS may be related to how much AGEs are accumulated in the body.In addition,studies by Qin L ect have shown that endoplasmic reticulum homeostasis can lead to cell autophagy and death,and the mechanism may be through negatively regulating the AKT / TSC / m TOR pathway to enhance cell autophagy.The abnormal increase of AGEs in diabetic patients may induce ERS,and enhance the autophagy of BMSCs cells by negatively regulating the AKT / TSC / m TOR pathway.Therefore,the role of AGEs on autophagy and osteogenic differentiation of BMSCs and its mechanism remain to be explored.This experiment is to study the effect of different concentrations of AGEs on autophagy during the osteogenic differentiation of rat BMSCs,and to provide new ideas for exploring the role of AGEs on autophagy in abnormal bone metabolism in diabetic patients.Purpose: The purpose of this experiment is to explore the effect of AGEs on the autophagy ability and osteogenic differentiation of BMSCs,explore its possible mechanisms,and provide new ideas for the treatment of osteoporosis in patients with diabetes.Methods: Part I: Different concentrations of AGEs were added to each group of BMSCs and cultured for 24 hours.Cell proliferation activity was detected by CCK-8.Transmission electron microscopy and immunofluorescence were used to detect the formation of autophagosomes.Real-time PCR and western blot techniques were used to detect autophagy-related genes in each group.The expression of beclin1 and LC3 were different.Then,the autophagy inhibitor 3-MA and the autophagy promoter rapamycin were added to the corresponding groups,and one week after osteogenesis induction,RT-q PCR and alkaline phosphatase staining were used to detect the autophagy and osteogenesis related groups.Gene expression.Part II: Different concentrations of AGEs were added to BMSCs and cultured for 24 h.The difference of endoplasmic reticulum stress and autophagosome formation in each group was detected by transmission electron microscope.The expression of ERK stress-related genes PERK,el F2α,and ATF4 in each group was detected by Real-time PCR.The ERS inhibitor GSK2606414 or PI3 K / AKT pathway inhibitor LY294002 was added to the AGEs100 mg / L and AHEs200 mg / L groups,and cultured for 24 hours.The autophagy-related factors beclin1 and LC3 were detected by RT-q PCR and Western blot Expression,total AKT and phosphorylated AKT protein expression detected by Western blot.Results: Part I: After adding AGEs at different concentrations to BMSCs for 24 hours,when AGEs were ≤100 mg / L,the CCK-8 test results showed that the proliferation activity of BMSCs increased after transmission of AGEs.Transmission electron microscopy and immunofluorescence showed that the number of autophagosomes increased and autophagy The expression of osteogenesis-related genes increased,and when AGEs ≥200 mg / L,the proliferation activity of BMSCs decreased after treatment with AGEs.Transmission electron microscopy and immunofluorescence showed that the number of autophagosomes decreased,and autophagy and osteogenesis-related gene expression decreased.Part II: After the MSCs of different concentrations were added to BMSCs for 24 hours,the ER stress increased with the increase of AGEs added.Real-time PCR results showed that the expression of ERS-related genes PERK,el F2α,and ATF4 increased.Compared with the AGEs 100mg / L(200mg / L)group,the expression of autophagy-related genes Beclin1 and LC3 was reduced in the AGEs 100 mg / L(200mg / L)group after adding ERS inhibitor GSK2606414 for 24 hours.Protein expression increased;ERS-related genes PERK,el F2α,ATF4 expression decreased.When the PI3 K / AKT pathway inhibitor LY294002 was added to the same group,compared with the AGEs 100 mg / L(200mg / L)group,the expression of cell autophagy-related genes Beclin1 and LC3 decreased;the expression of phosphorylated p-AKT protein decreased,and There was no significant difference in protein content,and there was no significant difference in m RNA expression of ERS-related genes PERK and el F2α.Conclusion: Different concentrations of AGEs have a two-way effect on autophagy and osteogenic differentiation of BMSCs,and one of the mechanisms may be due to the combined effect of AGEs on endoplasmic reticulum stress and PI3 K / AKT / m TOR pathway.