| Introduction: Diabetes mellitus is a group of chronic metabolic diseases characterized by long-term hyperglycemia caused by multiple etiologies.Type2 diabetes may lead to osteopenia,destruction of bone tissue microarchitecture,and increase of bone fragility through various pathways,leading to osteoporosis or osteoporotic fractures,but the specific mechanism is been not understood.In diabetic patients,hyperglycemia drives the accelerated formation and deposition of advanced glycation end-products(AGEs)in various systems and in the circulation,which can significantly inhibit the proliferation of osteoblasts and increase the activity of osteoclasts,ultimately leading to osteoporotic fractures.Bone marrow mesenchymal stem cells(BMSCs)have the potential of self-renewal and multi-directional differentiation,and are closely related to osteoporotic fractures.Accumulating evidence suggests that long noncoding RNAs(lnc RNAs)are involved in regulating the pyroptosis of BMSCs.The basic part of this study aims to investigate the effect of lnc RNA ORLNC1(NOMMUT016106.2)on the pyroptosis of BMSCs and the specific molecular mechanism under the stimulation of CML(the most common AGEs).The clinical part aims to explore the analysis of factors related to fracture risk in patients with type 2 diabetes,and to provide new theoretical basis and clinical evidence for the relationship between type 2 diabetes and fracture risk.The study specifically divided into the following three parts:Part One Effects of Lnc RNA ORLNC1 in pyroptosis of BMSCsObjective: In this part,we explore the biological effect of Lnc RNA ORLNC1 in pyroptosis of BMSCs.Methods:1.The mouse bone marrow mesenchymal stem cells were isolated and cultured and characterized by flow cytometry,adipogenic and osteogenic differentiation assays.2.BMSCs were treated with CML and then tested by TUNEL to verify the effect of CML treatment on pyroptosis.The expression levels of pyroptosis-related genes(NLRP3 and Caspase1)in BMSCs were detected by RT-q PCR,western blot and immunofluorescence.3.Change the expression of ORLNC1 in BMSCs by pc DNA3.1(Pc ORLNC1)and si RNA(si-ORLNC1),and combined with CML treatment to explore the effect of CML and ORLNC1 on pyroptosis of BMSCsResults:1.The mouse BMSCs were fibroblast-like,with granules and vacuoles.Flow cytometry showed that the BMSCs expressed CD73(+),CD90.2(+),CD11b(-),and CD34(-).After induction of osteogenic differentiation,calcified nodules were stained red with Alizarin Red dye,and after induction of adipogenic differentiation,globular lipids were stained red with Oil Red O dye.2.TUNEL showed that CML treatment could cause the BMSCs pyroptosis.The results of RT-q PCR and western blot indicated that the expression levels of NLRP3 and cl-Caspase1 were up-regulated,and the same results were obtained by immunofluorescence analysis.3.The expression of ORLC1 in the Pc ORLNC1 group was significantly increased.The expression of ORLC1 was also increased in BMSCs after CML treatment,but lower than Pc ORLNC1 group.The ORLC1 expression after combined CML treatment and si RNA was significantly lower than that after CML treatment alone.Both Western blot and immunofluorescence showed that CML up-regulated the expression of ORLNC1 to promote the pyroptosis of BMSCs,and knockdown of ORLNC1 significantly down-regulated the expression of pyroptosis-related proteins.Summary: CML promotes pyroptosis of BMSCs by upregulating the expression of ORLNC1.Part Two The mechanism of Lnc RNA ORLNC1 in pyroptosis of BMSCsObjective: In this part,we explore the mechanism of Lnc RNA ORLNC1 in pyroptosis of BMSCs.Methods:1.High expression of ORLNC1 was got after treatment of CML.We predicted the binding site of mi R-200b-3p and ORLNC1,and used the dual-luciferase reporter assay to verify it.The direct binding of ORLNC1 to mi R-200b-3p via Ago2 was detected by RIP-Pull-down,PCR and western blot.And the RT-q PCR was used to detect the expression levels of mi R-200b-3p after the altered expression of ORLNC1 and mi R-200b-3p,and the TUNEL method was used to detect the pyroptosis level of BMSCs.The expression levels of pyroptosis-related proteins were detected by western blot and immunofluorescence.2.We predict the binding site of mi R-200b-3p and foxo3,and verify it with dual-luciferase reporter assay.Western blot was used to detect the expression of Foxo3 protein after changing the expression of mi R-200b-3p and Foxo3,and the TUNEL was used to detect the pyroptosis of BMSCs.The expression levels of NLRP3 and Caspase1 were detected by western blot and immunofluorescence.3.According to ORLNC1 overexpression and knockdown in BMSCs,the BMSCs were randomly divided into five groups: NC,CML,CML+si-ORLNC1,CML+mi R-Mimic and CML+si-Foxo3 group.The expressions of ORLNC1,mi R-200b-3p and Foxo3 in each groups were detected by RT-q PCR and western blot.And the TUNEL,Western blot and immunofluorescence verified its effect.4.We verify the mechanism of Lnc RNA ORLNC1 in vivo experiment.After CML treatment,RT-q PCR and Western blot were arranged to analyze NLRP3 and Caspase1 in pyroptosis of BMSCs.Different levels of proteins expression were compared between CML treatment group and NC group.Results:1.The luciferase activity of ORLNC1-WT was significantly inhibited by mi R-200b-3p mimic and promoted by mi R-200b-3p inhibitor,both inhibitory/promoting effects were abolished by mutation of the possible binding site.The binding of ORLNC1 and mi R-200b-3p was confirmed by IP,Western blot and RT-q PCR.In the group of knockdown ORLNC1,mi R-200b-3p overexpression was dectected.As it shown by TUNEL staining,the overexpression of ORLNC1 promoted the pyroptosis of BMSCs,while mi R-200b-3p mimic alleviated it,this was also confirmed by the expression levels of NLRP3 and Caspase1 detected by WB and IP.2.Dual-luciferase reporter gene results showed that,with overexpression of Foxo3-WT,the fluorescence intensity of mi R-200b-3p mimic group was significantly lower than that of mi R-200b-3p inhibitor group,while no significant difference was detected in the Foxo3-mut group.Western blot results showed that the expression of mi R-200b-3p was significantly negatively correlated with Foxo3.The TUNEL staining results showed that overexpression of mi R-200b-3p inhibited cell pyroptosis,while up-regulation of Foxo3 and mi R-200b-3p promoted cell pyroptosis.In contrast,knockdown of mi R200b-3p alone promoted pyroptosis,while down regulation of both mi R200b-3p and Foxo3 alleviated pyroptosis.overexpression of mi R-200b-3p inhibit the level of NLRP3 and Caspase.3.The RT-q PCR and Western blot results showed that ORLNC1 was ce RNA of mi R-200b-3p,Both down-regulation of ORLNC1 and up-regulation of mi R-200b-3p can decrease the expression of Foxo3.No matter down-regulation of ORLNC1,up-regulation of mi R-200b-3p or downregulation of Foxo3 could alleviate CML-induced pyroptosis in BMSCs.4.In vivo experiments,the bone marrow mesenchymal stem cells of CML-treated mice were significantly destroyed.Compared with the NC group,the levels of ORLNC1 and Foxo3 were high and mi R-200b-3p was low in BMSCs of CML-treated mice.The expressions of cl-Caspase1 and NLRP3 were significantly up-regulated in BMSCs of CML-treated mice.Summary:1.ORLNC1 is ce RNA of mi R-200b-3p.2.Foxo3 is target gene of mi R-200b-3p.3.ORLNC1 promotes CML-induced pyroptosis of bone marrow mesenchymal stem cells by targeting the mi R200b-3p/Foxo3 pathway.Part Three Analysis of risk-related factors of fractures in patients with type 2 diabetes mellitusObjective: The purpose of this study was to investigate the correlation between diabetes-related factors and FRAX score in patients with type 2diabetes,and to provide a clinical trial basis for the study of the relationship between type 2 diabetes and fracture risk.Methods: 117 patients with type 2 diabetes in the Endocrinology Department of Shijiazhuang Second Hospital were selected(outpatients and inpatients,from January 2021 to December 2021,52 males and 65 females,aged 40-80 years).The age,gender,BMI,fracture history,parental hip fracture history,current smoking status,drinking status,history of adrenal corticosteroid medication,history of rheumatoid arthritis,history of other secondary osteoporosis,and,history of osteoporosis drugs such as bisphosphonates,calcitonin,PTH analogs,etc.patients were recorded separately.Fasting venous blood and random urine samples were taken from patients,automatic biochemical analyzer was used to measure fasting blood glucose(Fasting plasma glucose,FPG),glycosylated hemoglobin(Hb Alc),various blood lipid indexes,and urinary microalbumin and urinary microalbumin.Dual-energy X-ray absorptiometry was used to detect the bone mineral density of the left femoral neck of the patients,the FRAX evaluation system was used to evaluate the incidence of major osteoporotic fractures(MOF)and hip fractures(HF)in the next 10 years.Statistical analysis was performed using SPSS 21.0.Results:1.The MOF score of patients with type 2 diabetes was positively correlated with age,FBG,Hb A1 c,UACR levels,and had no relationship with the level of TC,TG,HDL-C,LDL-C,fasting insulin,and C-peptide.The HF score in patients suffered from type 2 diabetes was correlated with age,FBG,Hb A1 c,and UACR levels positively.Besides,there was no linear correlation between HF score and TC,TG,HDL-C,LDL-C,fasting insulin and C-peptide.2.FBG was positively correlated with MOF which was the FRAX score.The results of multiple linear regression analysis showed that: Model 1: FBG was positively correlated with MOF when using the FRAX score as the dependent variable,FBG as the independent variable,without adjusting other variables.Model 2: UACR as the independent variable,FBG was positively correlated with MOF.3.There was a positive correlation between FBG and HF(the FRAX score).Multiple linear regression analysis showed that: Model 1: With FRAX score as the dependent variable,FBG as the independent variable,FBG was positively correlated with HF.Model 2: UACR as the independent variable,FBG was positively correlated with HF.4.Hb A1 c was positively correlated with MOF(the FRAX score).The results of multiple linear regression analysis showed as following: Model 1:Taking MOF as the dependent variable and Hb A1 c as the independent variable,without adjusting other variables.Model 2: With MOF as the dependent variable,while Hb A1 c,and UACR as the independent variable,Hb A1 c was positively correlated with MOF.5.Multiple linear regression analysis showed that Hb A1 c was positively correlated with HF(the FRAX score)in the following models: Model 1: With HF as the dependent variable,Hb A1 c as the independent variable without adjusting other variable.Model 2: With HF as the dependent variable,while Hb A1 c,and UACR as the independent variable,Hb A1 c was positively correlated with HF.Summary:1.The increasing age,elevated FBG,Hb A1 c,and UACR levels in patients with type 2 diabetes were associated with the enhanced risk of major osteoporotic fractures and hip fractures,2.The blood glucose level in patients with type 2 diabetes was positively correlated with FRAX score.3.The Increasing of FBG and Hb A1 c indicates higher risk of fracture in type 2 diabetes patients.Conclusions:1.CML promotes pyroptosis of BMSCs by upregulating the expression of ORLNC1.2.ORLNC1 promotes CML-induced pyroptosis of bone marrow mesenchymal stem cells by targeting the mi R-200b-3p/Foxo3 pathway.3.The blood glucose level in patients with type 2 diabetes was positively correlated with the risk of bone fracture.Increasing of blood glucose level indicates higher risk of fracture in patients with type 2 diabetes. |
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