Effects Of Sclerostin And Irisin On Bones And Muscles In Postmenopausal Wome

Part 1 The influence of sclerostin and irisin on bone and muscle among postmenopausal womenObjectiveIn recent years,novel perspectives hold that bones and muscles act as endocrine organs because they can secret bone-derived cytokines and muscle-derived cytokines,playing roles in bone-muscle crosstalk,such as sclerostin and irisin,respectively.However,the influence of sclerostin and irisin on bone and muscle among postmenopausal women remains controversial,and few studies have reported the associations between these two cytokines and bone microarchitecture,morphometric vertebral fractures(MVFs),muscle mass,and strength in postmenopausal women.Based on the Beijing subgroup of the Chinese Vertebral Osteoporosis Study(ChiVos),this study aims to determine the prevalence and incidence of osteoporosis,osteoporotic fractures,and sarcopenia among postmenopausal women.Meanwhile,we also analyze the associations between serum sclerostin and irisin levels and parameters of bone and muscle among postmenopausal women,including osteoporotic fractures(e.g.MVFs),falls,bone mineral density(BMD),bone microarchitecture,muscle mass,and muscle function,etc.MethodsThis study enrolled community-dwelling postmenopausal women in Beijing of the Chi Vos,including a baseline survey(n=274)and a follow-up(n=139).The process of the two surveys is as follows:we collected general information,histories of osteoporotic fractures and falls,etc.by interviewer-administered questionnaire,measured anthropometric parameters(including height and weight),sampled fast blood for biochemical measurements,and evaluated muscle function by handgrip strength,timed up and go test(TUG),and short physical performance battery(SPPB).SPPB included the chair rising test(CRT).As for radiological assessment,participants accepted examinations of the lateral thoracic and lumbar spine X-rays,dual-energy X-ray absorptiometry,and high-resolution peripheral quantitative computed tomography,which could generate parameters of MVFs,BMD,body composition,and bone microarchitecture,etc.Through enzyme-linked immunosorbent assay(ELISA),the serum sclerostin level was measured in subjects participating in the baseline and the follow-up surveys,while the serum irisin level was detected in those who took part in the follow-up survey.Participants with data from both two surveys were included in the per-protocol set(PPS)for calculating the prevalence and incidence of osteoporosis,osteoporotic fractures,and sarcopenia.At the same time,we analyzed the association between the serum sclerostin level and parameters of bone and muscle according to the baseline survey,and the relationship between serum irisin level and parameters of bone and muscle from the follow-up survey.Results1.Postmenopausal women have a high prevalence of osteoporosis and osteoporotic fractures but a low prevalence of sarcopenia.In the PPS analysis,the median age of participants was 65.0 years old at baseline,and the median follow-up duration of the longitudinal cohort was 4.7 years.Among postmenopausal women,the prevalence of osteoporosis was 13.0%at baseline and 23.0%at follow-up,respectively,and the incidence of osteoporosis was 2.1%.At baseline,participants who had an osteoporotic fracture history accounted for 31.7%,and the prevalence of MVFs was 18.0%.At follow-up,the incidence of osteoporotic fractures and MVFs was 3.2%and 1.4%,respectively.The prevalence of sarcopenia among postmenopausal women was 4.0%at baseline and 3.2%at follow-up(p>0.009).2.The serum sclerostin level is highly associated with the prevalent MVFs,BMD,and bone microarchitecture among postmenopausal women.At baseline,the median of the serum sclerostin level was 109.2 pmol/L among all participants.The serum sclerostin level of participants with normal bone mass,osteopenia,and osteoporosis decreased significantly in turn,with medians of 127.96 pmol/L,106.53 pmol/L,and 76.28 pmol/L,respectively(p<0.001).No matter whether participants had an osteoporotic fracture history or fall history,the serum sclerostin level had no significant difference between the two groups.According to the quartiles of serum sclerostin level,all participants were separated into four groups.Compared to the lowest quartile of the serum sclerostin level group,the prevalence of MVFs was lower in the highest quartile of the serum sclerostin level group(odds ratio:0.34,95%confidence interval:0.14-0.85,p=0.021)without adjustment.However,serum sclerostin was not the independent factor for the prevalent MVFs after multivariate adjustment.The serum sclerostin level had positive correlations with areal BMD at the femoral neck,total hip,and lumbar vertebrae(rs=0.442～0.471,all p values<0.001),and volumetric BMD of total bone,trabecular bone,and cortical bone both at the distal radius and the distal tibia(rs=0.133～0.388,all p values<0.05).As for bone microarchitecture,the serum sclerostin level was positively associated with trabecular bone score(TBS)at the lumbar vertebrae(rs=0.194,p=0.002).Both at the distal radius and the distal tibia,the serum sclerostin level was positively correlated with trabecular bone volume fraction,trabecular number,and cortical thickness(rs=0.190～0.422,all p values<0.01),but negatively related to trabecular separation and trabecular inhomogeneity of Network(rs=-0.431～-0.404,all p values<0.001).In addition,the serum sclerostin level among postmenopausal women had no significant associations with appendicular lean mass(ALM),skeletal mass index(SMI),handgrip strength,the finish time of the CRT,and SPPB rank.3.The serum irisin level is related to fall risk,cortical porosity,and muscle strength in postmenopausal women.The cross-sectional results at the follow-up showed that the median serum irisin level was 3.91μg/ml among postmenopausal women.No matter whether participants had an osteoporotic fracture or fall history or MVFs,the serum irisin level had no significant difference between the two groups.According to whether the finish time of TUG was more than 12 seconds,participants were assigned into the "low fall risk" and "high fall risk"groups.The serum irisin level in postmenopausal women with high fall risk was significantly lower than those with low fall risk(2.22 μg/ml vs.4.16 μg/ml,p=0.024).Univariate linear regression analysis showed that the serum irisin level had positive associations with cortical bone volumetric BMD both at the distal radius and the distal tibia(radius:β=0.184,p=0.031;tibia:β=0.242,p=0.004),however,there were no significant associations after multivariate adjustment.As for bone microarchitecture,the serum irisin level was negatively correlated with cortical porosity both at the distal radius and the distal tibia.After adjusting by age,height,serum sclerostin level,and body fat ratio,only cortical porosity at the distal radius had a negative correlation with the serum irisin level(β=-0.276,p=0.003).The serum irisin level had positive associations with handgrip strength(rs=0.185,p=0.030)and SPPB rank(Kendall tau-b=0.146,p=0.034)in postmenopausal women,but it did not correlate with areal BMD,TBS,other bone microarchitecture parameters,ALM,and SMI.ConclusionIn conclusion,the prevalence of osteoporosis and osteoporotic fractures increases among postmenopausal women with aging,but the prevalence of sarcopenia is relatively low in this cohort.Higher sclerostin levels in postmenopausal women are associated with improved areal and volumetric BMD,bone microarchitecture,and a lower prevalence of MVFs,but the serum sclerostin level does not correlate with muscle mass and function.Postmenopausal women with a low serum irisin level have high cortical porosity,high fall risk,and weak muscle strength,but the serum irisin level has no associations with fracture or fall histories,BMD,trabecular bone microarchitecture,and muscle mass.Part 2 The mechanism of hydrogen peroxide impairs the osteogenesis of MC3T3-E1 cells by inducing pyroptosisObjectiveThe pathogenesis of osteoporosis includes decreased bone formation and increased bone resorption.Cell pyroptosis is one of the ways of regulated cell death,which is associated with the occurrence of osteoporosis.This study aims to investigate whether hydrogen peroxide(H2O2)can affect cell proliferation and osteogenic differentiation abilities of mouse embryo osteoblast precursor cells(MC3T3-E1 cells)by inducing cell pyroptosis.MethodsFirst,we determined the minimum H2O2 concentration that affects the proliferation of MC3T3-E1 cells by using a cell counting kit-8(CCK8).In the aspect of cell pyroptosis,we detected the relative mRNA expression of NLR family pyrin domain containing 3(Nlrp3),cysteinyl aspartate specific proteinase-1(Caspase-1),Caspase-11,Gasdermin D(Gsdmd),interleukin-1β(Il-1β),and Il-18 between the control and H2O2 groups by realtime quantitative polymerase chain reaction(RT-qPCR),and detected the protein level of Nlrp3,Caspase-1,Caspase-11,and Gsdmd by Western Blot.The level of Il-1β and Il-18 from cell protein and cell supernatant was measured by the ELISA between the control and H2O2 groups.As for osteogenic indicators,RT-qPCR was used to detect the transcriptional level of runt-related transcription factor 2(Runx2),collagen type I alpha 1 chain(Collal),and alkaline phosphatase(Alp)of undifferentiated MC3T3-E1 cells and osteogenic-differentiated MC3T3-E1 cells for five days between the control and H2O2 groups.In addition,on the 5th day of osteogenic differentiation,the Alp activity of the two groups was evaluated;on the 14th and the 21st days of osteogenic differentiation,the area of calcified nodules in both groups was evaluated by alizarin red staining.Results1.The 200 μM H2O2 can significantly reduce the proliferation of MC3T3-E1 cells.The result of CCK8 showed that the viability of MC3T3-E1 cells significantly decreased to 72.83%(p<0.05)after 24 hours of intervention with 200 μM H2O2.Therefore,the concentration of 200 μM was selected as the optimal H2O2 intervention concentration for subsequent experiments.2.The 200 μM H2O2 can induce pyroptosis in MC3T3-E1 cells through the canonical pathway.After MC3T3-E1 cells accepted 200 μM H2O2 intervention for 24 hours,the relative mRNA expression of pyroptosis indicators in the H2O2 group was significantly increased than those in the control group.Compared to the control group,the relative mRNA expression of Nlrp3,Caspase-1 in the canonical pathway,Caspase-11 in the non-canonical pathway,and Gsdmd in the H2O2 group increased by 98.3%,87.5%,44.4%,and 30.8%,respectively(all p values<0.05).As for protein expression,the level of Nlrp3,Pro Caspase1,Pro Caspase-11(50 KD),Pro Caspase-11(37 KD),and cleaved Gsdmd in the H2O2 group was significantly higher compared to the control group,which were 1.41 times,3.05 times,2.71 times,1.73 times,and 1.28 times of those in the control group,respectively(all p values<0.05).Compared to the control group,the expression of cleaved Caspase-1 was slightly elevated,which was 2.17 times of the control group,but there was no significant difference between the two groups(p=0.059).The expression of cleaved Caspase-11 and Gsdmd had no significant differences between the control and H2O2 groups.As for pyroptosis-specific inflammatory factors,the relative mRNA expression of Il-1β in the H2O2 group was slightly higher than that in the control group but without a significant difference(control group vs.H2O2 group:1.066±0.479 vs.1.527±0.603,p=0.359).However,the relative mRNA expression of Il-18 in the H2O2 group increased by 80.0%compared to the control group(p=0.004).The result of ELISA showed that both the level of Il-1β and Il-18 from cell protein in the H2O2 group were significantly higher than those in the control group,the level of Il-1β was 17.26±4.50 pg/mg and 26.01 ±4.26 pg/mg between the control and H2O2 groups(p=0.006),and the level of Il-18 was 90.84±5.70 pg/mg and 106.80±4.10 pg/mg between the two groups(p=0.004).In the cell supernatant,the secretion level of Il-18 in the H2O2 group was significantly higher than that in the control group(control group vs.H2O2 group:19.53±2.88 pg/ml vs.23.79±1.91 pg/ml,p=0.048).3.The 200 μM H2O2 can reduce the osteogenic differentiation ability of MC3T3-E1 cells.After MC3T3-E1 cells accepted 200 μM H2O2 intervention for 24 hours,in the undifferentiated MC3T3-E1 cells,compared to the control group,the relative mRNA expression of Collal and Runx2 was significantly decreased by 31.1%(p=0.001)and 17.6%(p=0.007),while the relative mRNA expression of Alp was significantly increased by 1.79 times in the H2O2 group.On the 5th day of osteogenic differentiation,compared to the control group,only the relative mRNA expression of Col1a1 was significantly decreased by 48.1%(p=0.003)in the H2O2 group,while the relative mRNA expression of Runx2 and Alp was significantly increased by 81.9%(p<0.001)and 4.47 times(p=0.001).Moreover,on the 5th day of osteogenic differentiation,the Alp activity in the H2O2 group was significantly decreased,which was 3.21±0.29 DEA/g in the control group and 1.70±0.14 DEA/g in the H2O2 group,respectively(p<0.001).On the 14th day of osteogenic differentiation,the area of calcified nodules was 0.617±0.055 mm2 in the control group and 0.111±0.047 mm2 in the H2O2 group,respectively(p<0.001);when on the 21st day of osteogenic differentiation,the area of calcified nodules in the H2O2 group was also significantly decreased than that in the control group(control group vs.H2O2 group:0.562±0.069 mm2 vs.0.394±0.080 mm2,p<0.001).ConclusionH2O2 can inhibit the proliferation of MC3T3-E1 cells and reduce their osteogenic differentiation ability by activating the canonical pathway of cell pyroptosis.Inhibiting pyroptosis of osteoblasts may be a potential therapeutic direction for osteoporosis.