| Objective: Epidemiology suggests that children exposed to adverse environments during pregnancy are susceptible to hypercholesterolemia in adulthood,and its pathogenesis has an intrauterine origin.Dexamethasone,as a synthetic glucocorticoid,has been widely used in the clinic for the treatment of preterm labor.Epidemiology suggests that prenatal dexamethasone abuse will lead to the offspring's long-term susceptibility to diseases such as obesity,nervous system,osteoarthritis and metabolic syndrome.Although some animal studies have found that prenatal dexamethasone exposure(PDE)can raise blood cholesterol in offspring after birth,whether it has an intrauterine origin phenomenon and its intrauterine programming mechanism has not been reported.The purpose of this study was to confirm the effect of PDE on hypercholesterolemia in adult offspring of male rats and to elucidate the epigenetic programming mechanism in utero.Methods: Wistar rats pregnant after 9~21 days(gestational day 9~21,GD9~21)neck subcutaneous injection of dexamethasone(0,0.1,0.2 and 0.4 mg/kg.d),part of the pregnant rats born(GD21)after anesthesia to take blood and fetal liver tissue,part through natural birth in different period(PW12 and PW28)executed after adult,take blood and liver tissue,at the moment for F1 generation,again through maternal genetic F2 and F3 generation also executed in PW12 weeks,in the male rat blood and liver tissue.Serum cholesterol levels before and after birth were determined by enzymatic biochemical reagents.The m RNA and protein expression levels of multiple genes in multi-generation liver tissues were detected by real-time quantitative PCR and western blotting,including glucocorticoid receptor(GR),3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR),apolipoprotein B(Apo B),scavenger receptor B1(SR-B1),low-density lipoprotein receptor(LDLR),Di George syndrome critical region 8 gene(DGCR8),helicase with RNase motif,Dicer)and 2ribonuclease III enzymes(Class 2 ribonuclease III enzym,Drosha).Enhanced transcriptional regulation of GR on the DGCR8 promoter region was detected by chromatin immunoprecipitation.Epigenetic regulatory mechanisms were screened by mi RNA extraction and detection.At the same time,the effect of dexamethasone on the liver in the process of development and differentiation was first simulated using the hepatolike differentiation model of bone marrow mesenchymal stem cells(BMSCs),and the effect of dexamethasone on the expression of cholesterol-related metabolic genes and the epigenetic mechanism of regulation of LDLR were verified on the hepatolike differentiation model of BMSC and liver cell lines.Results: intrauterine period: compared with the control group,fetal blood TCH in the PDE group was dose-dependent(P<0.05),while HDL-C and LDL-C levels were significantly reduced(P<0.05).Furthermore,it was found that the contents of TCH in the fetal liver were dose-dependent(P<0.05,P<0.01),and the gene and protein expressions of cholesterol metabolism-related genes(HMGCR,Apo B,SR-B1,LDLR)in the fetal liver were decreased in both the PDE(M)group and the PDE(H)group(P<0.05).Compared with the control group,the m RNA expression of GR in fetal liver in the PDE group increased,and the expression of GR in fetal hepatic nuclein increased(P<0.01).The m RNA expression levels of mi RNA synthesis recognition enzyme DGCR8 and mi RNA shear enzyme Dicer in fetal liver were significantly increased(P<0.05),and the expression levels of mi R-148 a were significantly increased(P<0.05).After birth: compared with the control group,PD1 body weight in the PDE(M)group decreased significantly(P<0.05,P<0.01),while that in the PW2-6 group increased significantly,PW7-12 was close to the control group,and the weight growth rate of PD1-PW12 was always significantly higher than that in the control group(P<0.01).After that,changes in serum cholesterol levels were detected.Compared with the control group,serum TCH levels of PW12 and PW28 in the PDE group increased(P<0.05,P<0.01),while HDL-C level increased in PW12 and decreased in PW28(P<0.05,P<0.01).LDL-C level did not change significantly in PW12 but increased in PW28(P=0.055).The results of this study showed that the blood TCH/ HDL-C and LDL-C/HDL-C ratios of PW12 and PW28 in the PDE group increased(P<0.05).The results showed that the contents of TCH in PW12 and PW28 liver tissues increased(P<0.05).At PW12,the m RNA and protein levels of the liver cholesterol output gene Apo B increased in the PDE group(P<0.05),and the m RNA and protein levels of the cholesterol reversal genes SR-B1 and LDLR decreased(P<0.05),while the m RNA and protein levels of the cholesterol synthesis function gene HMGCR did not significantly change.At PW28,the m RNA and protein levels of HMGCR,Apo B and LDLR in the PDE group decreased(P<0.05,P<0.01),while the m RNA and protein levels of SR-B1increased(P<0.05,).In F2 and F3 multiple-generation of research,found that compared to the control group,serum cholesterol,liver mi R-148 a expression and LDLR m RNA expression in the PDE group were not significantly changed in F2 generation,and the SR-B1 m RNA level increased significantly(P<0.05),and cholesterol levels in the blood of the PDE group F3 generation increases,the liver mi R-148 a expression increased,and the LDLR m RNA expression reduced(P<0.05,P<0.01).Cell assay: by dil-ac-ldl detection and PAS staining,it was found that there were concentrations of low-density lipoprotein,glycogen and ALB in the differentiated hepatoid cells of BMSC.Flow cytometry identified the proportion of ALB labeled cells in 93.8% and CK18 labeled cells in 84.2%.These results suggested that the differentiation of BMSCs hepatoid cells was successful.BMSCs hepatoid cells showed that after dexamethasone treatment,the level of TCH in the extracellular supernatant was not only time-dependent(P<0.01),but also concentration-dependent(P<0.05,P<0.01).Meanwhile,the downstream target gene Sgk1 of GR was increased in concentration dependence(P<0.01),indicating that GR was activated.It was further found that the expressions of DGCR8,Dicer and mir-148 a were increased in concentration dependence(P<0.05,P<0.01),and the expressions of LDLR m RNA and protein levels were decreased in concentration dependence(P<0.05,P<0.01).Interference with LDLR,mir-148 a and GR on Hep G2 cells revealed that dexamethasone could significantly increase the intracellular and extracellular cholesterol levels in the extracellular high-cholesterol environment(P<0.01),while overexpression of LDLR could reverse the extracellular cholesterol level increase caused by dexamethasone(P<0.01),and further increase the intracellular cholesterol level(P<0.05).mi R-148 a inhibitor can significantly reverse the decrease of LDLR expression caused by dexamethasone(P<0.05,P<0.01),and also at a certain extent to reverse the increase of extracellular cholesterol caused by dexamethasone,and increase the amount of cholesterol transported to the intracellular level(P<0.05,P<0.01).GR inhibitor RU486 can significantly reverse the increased m RNA expression of DGCR8 and mi R-148 a caused by dexamethasone,and the m RNA and protein expression of LDLR(P<0.05,P<0.01).We performed Ch IP-PCR detection on Hep G2 cells and confirmed that the DGCR8 promoter region was increased with GR after dexamethasone treatment(P<0.01).Conclusion: the PDE can cause adult male offspring rats hypercholesterolemia,it mainly through the activation of hepatic GR enhance DGCR8 intrauterine dexamethasone and mi R-148 a express,and mi R-148 a high expression from intrauterine continue into adult F3 generations,even programming LDLR continued low expression,reverse cholesterol transport lead to liver function damage,eventually lead to high blood cholesterol. |
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