Mechanism And Clinical Value Of Type 2 Diabetes Mellitus With Sarcopenia

Part One Changes and clinical significance of inflammatory indicators for type 2 diabetes mellitus with sarcopenia in middle-aged and elderly patientsObjective:Patients with type 2 diabetes mellitus(T2DM)increase the risk of sarcopenia.Chronic inflammation is involved in the pathogenesis of both.This part of the study aims to explore the changes and clinical significance of NLRP3 and inflammatory complex indicators of T2DM with sarcopenia in middle-aged and elderly patients.Method:The patients with T2DM hospitalized in the Department of Endocrinology of the First Hospital of Qinhuangdao from October 2019 to September 2022 were collected in this study.Dual-energy X-ray was used to determine the body composition.The appendicular skeletal muscle mass index(ASMI)of the limbs was defined as the skeletal muscle mass(kg)of the limbs divided by the square of the height(m).The determination of grip strength,and the evaluation of the 6-meter walking speed were completed.Serum samples were collected and biochemical indicators were measured.NLRP3,IL-1βand IL-8 inflammatory indicators were measured.Inflammatory complex markers including the neutrophil to lymphocyte ratio(NLR),platelet to lymphocyte ratio(PLR),systemic immune inflammation index(SII),and prognostic nutritional index(PNI)were calculated.A total of 448 people were enrolled.According to the presence or absence of sarcopenia,patients were divided into sarcopenia group(152 cases)and non-sarcopenia group(296cases).Results:1.Compared with non-sarcopenia group,the mean age of T2DM patients in sarcopenia group was higher[68.8(8.79)vs 64.4(8.41),P<0.05)],the duration of diabetes was longer[11.7(9.59)vs 9.6(8.10),P<0.05],and the incidence rate of cardiovascular disease was higher(28.9%vs 19.6%,P<0.05).BMI levels[24.26(2.86)vs 25.99(3.24)kg/m~2],vitamin D levels[42.07(11.22)vs 46.40(13.97)nmol/L]and plasma albumin levels[41.54(4.8)vs 43.79(13.97)g/L]in sarcopenia group were lower compared with those in non-sarcopenia group.The difference was statistically significant(P<0.05).2.Compared with non-sarcopenia group,NLRP3[5.92(0.82)vs 5.52(0.74)],IL-1β[2.57(0.54)vs 2.18(0.42)],IL-8[4.77(0.96)vs 4.50(0.78)]and neutrophil[3.97(1.43)vs3.68(1.33)]were higher in the sarcopenia group with statistically significance(P<0.05).In the evaluation of systemic inflammation,PNI was decreased in the sarcopenia group and the difference was statistically significant(P<0.05).3.Correlation analysis between ASMI and other variables showed that ASMI was negatively correlated with age(r=-0.112),Hb A1c(r=-0.097),duration of diabetes(r=-0.105),NLRP3(r=-0.120)and IL-1β(r=-0.148).The difference was statistically significant(P<0.05).ASMI was positively correlated with muscle mass(r=0.808),BMI(r=0.403),vitamin D(r=0.276),plasma albumin(r=0.105)and PNI(r=0.102),and the differences were statistically significant(P<0.05).4.NLRP3 and IL-1βwere negatively correlated with ASMI,grip strength and step speed,with statistically significance(P<0.05).IL-8 was negatively correlated with step speed and difference was statistically significant(P<0.05).PNI was positively correlated with ASMI,grip strength and gait speed,and the differences were statistically significant(P<0.05).5.Multiple linear regression analysis showed that ASMI as the dependent variable,age,Hb A1c,duration of diabetes,NLRP3,IL-1β,vitamin D,plasma albumin,BMI and PNI were the independent variables.Vitamin D level(β=0.237,P<0.05)and BMI(β=0.378,P<0.05)were positively with ASMI.Hb A1c(β=-0.097,P<0.05)and IL-1β(β=-0.184,P<0.05)was negatively with ASMI(P<0.05).6.Multiple logistic regression analysis showed that sarcopenia as the dependent variable,NLRP3,IL-1β,IL-8,PNI,gender,age,BMI,diabetes course,Hb A1c,coronary heart disease history,plasma albumin and vitamin D levels were as the independent variables.The risk of male with sarcopenia was increased by 85%(OR=1.847,95%CI 1.016～3.085).High level of NLRP3(above P50th)(OR=2.047,95%CI 1.234～3.396),high level of IL-1β(above P50th)(OR=2.087,95%CI 1.254～3.473)and people with cardiovascular disease(OR=2.257,95%CI 1.284～3.967)increased risk of sarcopenia(P<0.05).Plasma albumin(OR=0.943,95%CI 0.898～0.991),vitamin D(OR=0.968,95%CI 0.948～0.989)and BMI(OR=0.802,95%CI 0.735～0.875)all reduced the risk of sarcopenia(P<0.05).Conclusions:1.Patients with T2DM in sarcopenia group had a higher mean age,a longer history of diabetes,a higher incidence of cardiovascular diseases,and a lower level of BMI,vitamin D and plasma albumin compared with non-sarcopenia group.2.Compared with non-sarcopenia group,the inflammatory markers NLRP3,IL-1β,IL-8 and neutrophil counts were increased and PNI was decreased in T2DM patients with sarcopenia.3.In multiple logistic regression analysis,males,high levels of NLRP3and IL-1β,and cardiovascular disease were independent risk factors for sarcopenia in T2DM patients.The higher the level of plasma albumin,vitamin D and BMI,the lower the risk of sarcopenia in T2DM patients.Part Two Role of autophagy-NLRP3 in hyperglycemia induced senescence of mouse C2C12 muscle cellsObjective:To verify whether high glucose can affect the senescence through autophagy-NLRP3 inflammasome,we intervened C2C12 cells with different glucose concentrations,exploring the potential pathogenesis of T2DM with sarcopenia.Method:1.Cultured C2C12 muscle cells were stimulated with different concen-trations of glucose(5.5m M,25m M,40m M)and transfection with si RNA-NLRP3.The expression of NLRP3 inflammasome-related proteins,MURF1 and Myogenin were detected by WB and immunofluorescence.IL-1βlevels in cell culture medium were detected by Elisa.2.C2C12 cells were divided into different intervention groups[Control group(5.5m M/L glucose),High Glucose(40m M/L glucose),Control+3-Methyladenine(MA)(5m M),High Glucose+Rapamycin(RA)(200n M)]to explore the effects of autophagy on inflammatory activation and senescence.Mcherry-EGFP-LC3 labered adenovirus were transfected.And the autophagosome formation process was detected by laser confocal and electron microscopy techniques.The expression levels of autophagy associated proteins,NLRP3 inflammasome associated proteins,MURF1 and Myogenin were detected by WB and immunofluorescence.IL-1βlevels in cell culture were detected by Elisa.Results:1.Compared with Control group,the proteins expression of Caspase-1,NLRP3 and MURF1,the immunofluorescence intensity of ASC and the activity of IL-1βin cell culture medium increased,while the proteins expression of Myogenin decreased in High Glucose-25m M group and High Glucose-40m M group(P<0.05).Compared with High Glucose-40m M+NC group,the protein expression of Caspase-1,NLRP3 and MURF1 decreased,while Myogenin increased in High Glucose-40m M+Si NLRP3 group.The immunofluorescence intensity of ASC decreased,and the activity of IL-1βin culture medium decreased in High Glucose-40m M+Si NLRP3 group compared with High Glucose-40m M+NC group(P<0.05).2.The results of autophagy double labeled adenovirus detection showed that compared with Control group,the number of autophagosome and autophagylysosome decreased in High Glucose group and Control+3-MA group,and which was increased in High Glucose+RA group compared with High Glucose group.The differences were statistically significant(P<0.05).Compared with Control group,p62 protein expression increased,LC3II/I ratio decreased and p62 immunofluorescence level increased in High Glucose group and Control+3-MA group.Compared with High Glucose group,p62protein expression decreased,LC3II/I ratio increased and p62immunofluorescence level decreased in High Glucose+RA group.The differences were statistically significant(P<0.05).3.Compared with Control group,the activity of IL-1βincreased,the protein levels of Caspase-1,NLRP3 and MURF1 increased,the protein level of Myogenin decreased,and the immunofluorescence intensity of ASC increased in High Glucose group and Control+3-MA group.Compared with High Glucose group,IL-1βactivity decreased,Caspase-1,NLRP3 and MURF1 protein levels decreased,Myogenin protein level increased and ASC immunofluorescence intensity decreased in High Glucose+RA group.The differences were statistically significant(P<0.05).Conclusions:High glucose induces the activation of NLRP3inflammasomes in C2C12 muscle cells in a dose-dependent manner,elevates the level of IL-1βand promotes the inflammatory response.High glucose also leads to the increased expression of ubiquitination-related MURF1 protein,and promotes the occurrence of cell senescence.The above process is related to the decrease of autophagy flux in C2C12 muscle cells.Appropriate enhancement of autophagy can reduce the level of inflammatory response in muscle cells and alleviate the occurrence of cell senescence.Part Three Erector spinal muscle index as a prognostic marker for type 2 diabetes mellitus with community-acquired pneumoniaObjective:Patients with T2DM are at high risk of infection and are prone to severe pneumonia.The assessment of skeletal muscle mass and function is of great significance for judging the nutritional status and survival prognosis.Skeletal muscle mass is significantly reduced in patients with T2DM,further increasing the risk of death from infection.In this study,erector spinal muscle index related to 12 thoracic vertebrae were used as a convenient and quick way to evaluate human muscle mass.We aimed at exploring its impact on prognosis and long-term survival of T2DM patients with community-acquired pneumonia(CAP),identifying early risk and taking intervention for patients with poor prognosis.Method:A total of 696 patients with T2DM who were hospitalized for CAP in the First Hospital of Qinhuangdao from January 2015 to December2018 were retrospectively selected,including 400 males and 296 females,with an average age of 71.6±11.7 years.The basic information of gender,age,length of hospitalization,and hospitalization cost were collected.Diabetes,medical history,diseases associated with chronic kidney disease and heart failure of patients were collected.The occurrence of septic shock,whether ventilator was used and whether death occurred after discharge were recorded.And the lung imaging data of patients were collected.Bilateral erector spinalis(dorsal skeletal muscle area at T12 vertebral level,T12MA)was defined as any muscle in the posterior area of the spine and ribs of the T12 that does not extend beyond the outermost edge of the erector spinalis.Skeletal muscle index(SMI)was defined as the ratio of T12MA to height(m)squared.According to whether the discharged patients died or not,they were divided into survival group(655 cases)and death group(41 cases).Results:1.Compared with the survival group,the mean age of patients in the death group was higher[78.0(11.7)vs71.2(11.6)],and the proportions of patients with heart failure(53.7%vs 23.5%)and chronic kidney disease(22.0%vs10.7%)were higher.In the death group,the incidence of invasive ventilation(19.5%vs 3.2%),septic shock(19.5%vs 2.9%)and ICU hospitalization(39.0%vs 12.7%)were higher than those in the survival group.The difference was statistically significant(P<0.05).Patients in the death group had high white blood cell count[14.8(13.1)vs 9.8(5.9)]and high CURB65 score(2vs 1).And the plasma albumin level[30.7(5.1)vs 35.6(11.3)g/L],BMI[22.0(3.9)vs 24.9(4.5)kg/m~2],T12MA[27.9(8.75)vs 33.8(12.9)cm~2]and SMI[9.99(2.95)vs 12.4(3.4)cm~2/m~2]values were lower than those of the survival group,with statistically significance(P<0.05).2.Correlation analysis showed that SMI was negatively correlated with age(r=-0.359,P<0.05),CURB65(r=-0.295,P<0.05)and WBC(r=-0.095,P<0.05),but positively correlated with plasma albumin(r=0.122,P<0.05)and BMI(r=0.483,P<0.05).3.Multiple logistic regression analysis showed that death of hospitalized patients as the dependent variable,gender,age,smoking history,BMI,T12MA,SMI,chronic kidney disease,heart failure,WBC,CURB65 and plasma albumin were the independent variables.SMI(OR=0.876,95%CI:0.768～0.998,P<0.05)and plasma albumin level(OR=0.894,95%CI:0.883～0.959,P<0.05)were independent protective factors for death in T2DM patients with CAP.CURB65(OR=1.630,95%CI:1.077～2.466,P<0.05)and heart failure(OR=6.250,95%CI:2.721～14.360,P<0.05)were independent risk factors.Propensity score matching(PSM)was used to reduce the confounding offset.The nearest neighbor matching method was used to match the death group and the survival group at 1:3.After matching,logistic regression analysis showed that SMI was an independent protective factor(OR=0.849,95%CI:0.738～0.976,P<0.05),while heart failure(OR=2.772,95%CI:1.262～6.093,P<0.05)and WBC(OR=1.109,95%CI:1.034～1.190,P<0.05)were independent risk factors for death in T2DM patients complicated with CAP.4.COX regression analysis of the long-term survival of T2DM patients with CAP showed that death at the follow-up cut-off point as the dependent variable.T12MA,SMI,age,sex,smoking history,BMI,plasma albumin level,CURB65 score,chronic kidney disease,heart failure,coronary atherosclerotic heart disease,cerebrovascular disease,septic shock and ICU were independent variables.SMI was an independent protective factor for long-term death in T2DM patients with CAP(HR=0.929,95%CI:0.884～0.976,P<0.05).Heart failure(HR=1.600,95%CI:1.129～2.268,P<0.05),coronary atherosclerotic heart disease(HR=1.488,95%CI:1.059～2.090,P<0.05)and CURB65(HR=1.585,95%CI:1.346～1.866,P<0.05)were independent risk factors for long-term prognosis of T2DM with CAP.After matching,COX regression analysis showed that SMI was an independent protective factor(HR=0.880,95%CI:0.816～0.949,P<0.05),while heart failure(HR=2.420,95%CI:1.593～3.677,P<0.05)and CURB65(HR=1.327,95%CI:1.051～1.675,P<0.05)were independent risk factors for long-term death in T2DM patients complicated with CAP.Conclusions:In the death group of T2DM patients with CAP,the older the age,the higher the risk of death.Among them,the proportion of patients with heart failure,chronic kidney disease,invasive ventilation,septic shock,and ICU hospitalization was higher.SMI levels were independent protective factors for death in hospitalized T2DM patients with CAP.Heart failure was independent risk factors for death in hospitalized patients.SMI can affect in-hospital death and long-term survival in T2DM patients with CAP.