Study On Ameliorating Osteoporosis Of Type 2 Diabetes Mellitus By Malvidin Regulating NIPA2 And Its Mechanism

Objective: Diabetes osteoporosis(DOP)is a systemic osteopathy characterized by low bone mass,destruction of bone microstructure,increased bone fragility,and fracture prone,which is complicated on the basis of diabetes.The risk of secondary osteoporosis and brittle fracture in diabetes patients was significantly higher than that in the general population,and the incidence rate of DOP in type 2 diabetes patients was as high as 60%.Such a high incidence rate has caused a great burden to the medical resources and socioeconomic around the world.Therefore,it is of great social significance to effectively prevent and control DOP and reduce its incidence rate.Food rich in anthocyanins has a potential role in the prevention and treatment of chronic diseases such as diabetes.Population studies found that regular consumption of vegetables and fruits rich in anthocyanins can effectively prevent the loss of bone minerals,reduce the risk of osteoporosis and promote fracture healing.The intake of anthocyanins is positively correlated with the bone mineral density of the study population.Previous studies have proved that feeding anthocyanin to ovariectomized rats or mice can resist bone loss caused by ovariectomy,and thus have a certain protective effect on osteoporosis.However,there are few reports on the animal model of diabetes induced osteoporosis.Only domestic scholar Qi and others reported that feeding anthocyanin rich black rice extract can improve the bone loss caused by diabetes by establishing a diabetes rat model.Therefore,the experimental evidence and theoretical mechanism of anthocyanins in improving diabetes induced osteoporosis are still insufficient.This subject selected malvidin,the highest component content of blueberry anthocyanins as the drug,designed in vivo and in vitro experiments to further verify the effect of anthocyanins on diabetes induced osteoporosis,explore its biological regulation mechanism,and find new therapeutic targets.Methods: 1、Type 2 diabetes model db/db mice and control model db/m mice with the same background were randomly divided into control group(CC),diabetes model group(DM),low-dose M3 G treatment group(LM)and high-dose M3 G treatment group(HM).Fasting blood glucose(FBG)was measured by a glucose meter;The radioimmunoassay kit was used to detect fasting insulin(FINS);Serum BALP and TRAP5 b levels were measured by ELISA;Micro-CT scanning was used to analyze the microstructure of tibia and bone in mice,parameters: BMD,BV/TV,Tb.N,Tb.Th and Tb.Sp;Preparation of nondecalcified bone sections and Masson-Golder Trichrome staining,bone histomorphometric analysis,parameters: N.Ob/T.Ar,Ob.S/BS,MAR,BFR/BS;Three-point bending test of femur and biomechanical analysis of bone,parameters: maximum load,stiffness;RT-PCR and Western Blot were used to detect the expression of osteogenic related factors(Runx2,BMP2,OCN,OPG)in femur tissue.2.The content of magnesium in femur was determined by flame atomic absorption spectrometry;The expression of NIPA2 was evaluated by immunohistochemistry;In vitro experiment: MC3T3-E1 cells were divided into control group(Control),high glucose group(HG),low concentration of malvidin group(L-M3G)and high concentration of malvidin group(H-M3G).RT-PCR and Western blot were used to detect the m RNA and protein expression of NIPA2 in cells of each groups;Mag-Fura-2 AM magnesium ion fluorescence probe was used to observe the expression of magnesium ion in cells in each group;Construction of silent and overexpression NIPA2 lentivirus,transfection of MC3T3-E1 and screening of stable expression cell lines,re-grouping: control group,M3 G group,M3G+LV-Ctrl-RNAi group,M3G+LV-NIPA2-RNAi group,M3G+LV-Ctrl group and M3G+LV-NIPA2 group,the following experiments were carried out: The m RNA expression of osteogenic related factors(ALP,OCN and OPG)was detected by RT-PCR;Alizarin red calcium nodule staining was used to evaluate the calcium nodule density of cells in each group;The osteoblastic activity of the cells in each group was evaluated by alkaline phosphatase staining.3.Magnesium ions fluorescence probe was used to verify that M3 G promoted the inflow of magnesium ions in MC3T3-E1 cells by regulating the expression of NIPA2;The expression of autophagy associated proteins(LC3,Beclin1,Atg3,p62)was detected by Western blot;The expression of LC3 protein was detected by immunofluorescence;The formation of autophage was observed by transmission electron microscope;Western blot method was used to detect the effect of autophagy regulator on M3G’s regulation of osteoblast autophagy(LC3,p62)and osteogenic function(OCN,OPG)through NIPA2,the participation of PI3K/AKT/m TOR signal pathway and the influence of regulating PI3K/AKT/m TOR signal pathway on M3G’s regulation of osteoblast autophagy through NIPA2.Results: 1.M3 G improved blood glucose and insulin resistance in db/db mice.Serological indicators suggest that M3 G can improve the bone formation level and reduce the bone absorption level in db/db mice.Micro-CT scan showed that M3 G significantly improved the trabecular bone microstructure of db/db mice.The morphometric analysis showed that M3 G could increase the number and activity of osteoblasts in the tibia of db/db mice,and accelerate the rate of bone mineralization and bone formation.The biomechanical analysis of femur suggested that M3 G could improve the biomechanical properties of the femur of db/db mice,and improve the total mechanical strength and fracture toughness of the femur of db/db mice.Femoral RT-PCR and Western Blot showed that M3 G increased the m RNA and protein expression of osteogenic related factors(Runx2,BMP2,OCN and OPG)in the femur of db/db mice.2.M3 G can increase the content of magnesium in the femur of db/db mice and the expression of NIPA2 protein in the tibia.In vitro,M3 G increased the expression level of NIPA2 protein and m RNA in MC3T3-E1 cells,and then increased the level of magnesium ion in cells.By up-regulating and silencing NIPA2,it is further proved that M3 G can improve the expression of osteogenic factors(ALP,OCN and OPG)m RNA in MC3T3-E1 cells through positive regulation of NIPA2,increase the number of calcium nodules stained by alizarin red,and increase the number and coloring depth of cells with positive reaction of alkaline phosphatase.3.M3 G can regulate magnesium ion flow in MC3T3-E1 cells through NIPA2.NIPA2 can inhibit the autophagy level of MC3T3-E1 cells,and then affect the expression of autophagy-related proteins.The results of immunofluorescence detection and transmission electron microscopy of LC3 protein were consistent with those of Western blot.The introduction of RAPA and 3-MA again confirmed that M3 G could inhibit the autophagy of MC3T3-E1 cells by increasing the expression of NIPA2,thereby improving the osteogenic function of the cells.This autophagy is excessive autophagy in the pathological state of diabetes osteoporosis.M3 G increased the phosphorylation level of PI3 K,AKT and m TOR,and the phosphorylation level was further increased after overexpression of NIPA2,which proved that the PI3K/AKT/m TOR signal pathway was involved in the regulation of autophagy of osteoblasts by M3 G through NIPA2.Moreover,activation or inhibition of this pathway can increase the phosphorylation level of PI3 K and then inhibit or activate autophagy.Conclusion: 1.The application of M3 G in the treatment of db/db mice by gavage can significantly observe the improvement of serum bone metabolism,bone density and microstructure of the mice,morphological and quantitative parameters of the tibia,and structural characteristics of the femur,as well as the expression of osteogenic related factors in the femur.It is concluded that M3 G can improve osteoporosis in db/db mice by regulating the expression of osteogenic related factors.2.In vivo experiments have shown that M3 G gavage treatment can increase the expression of NIPA2 in bone tissue of db/db mice,thereby increasing magnesium content levels.In vitro experiments have shown that M3 G can positively regulate the expression of NIPA2,increase magnesium ion levels in osteoblasts,promote osteoblast proliferation and mineralization,and improve osteogenic function.3.By positively regulating the expression of NIPA2,M3 G promoted the inflow of magnesium ions in MC3T3-E1 cells,and further regulated the autophagy of cells,thus affecting the osteoblastic function of the cells.The specific mechanism may be that M3 G can promote the magnesium influx and activate the PI3K/AKT/m TOR signal pathway by increasing the expression of NIPA2,thereby inhibiting the autophagy of osteoblasts,promoting the expression of osteogenic related factors in cells,and improving the osteogenic function of cells.