| Objective:Diabetic retinopathy(DR)is one of the common complications of diabetes.Its early characteristic pathological changes are apoptosis of retinal microvascular cells caused by hyperglycemia.Studies have shown that serum thyroid stimulating hormone(TSH)levels may be one of the independent risk factors for DR,but this view is still controversial,and its mechanism has not been elucidated.The purpose of this study is to explore the correlation between TSH and DR,and to clarify its potential mechanism.Methods:In order to explore the relationship between TSH and DR and its mechanism,this study intends to be carried out from the holistic,cellular and molecular level,respectively.Part One Clinical study1.Human subjects:The population in this study was patients with type 2 diabetes mellitus(T2DM)who were hospitalized in the Department of Endocrinology and Metabolism of Beijing Hospital from January 2018 to July 2019.2.Data collection:We collected clinical data of 1,121 patients,including their gender,age,medical history,medication history,etc.,as well as related auxiliary examine,such as body mass index(BMI),blood lipids,blood glucose,blood pressure,thyroid function,glycosylated hemoglobin(HbAlc),fundus imaging,etc.A total of 365 DR patients and 633 non-DR patients were included according to the inclusion criteria.3.Statistical analysis:3.1 According to with DR or not,the subjects were divided into 2 groups.Non-normal Mann-Whitney u test was used to compare the differences between the continuous variables in baseline clinical data between the two groups;chi-square test was used to compare the differences between the binary variables between the two groups.Two-sided test p<0.05 can be considered statistically significant difference between groups.3.2 According to the level of serum TSH,the subjects were divided into 4 groups,namely 0-0.35?IU/ml;0.35-2.49 ? IU/ml;2.49-5.55 ?IU/ml and>5.55 ? IU/ml group.Chi-square test was used to analyze whether the TSH level of each group was related to the prevalence of DR and the severity of DR.Two-sided test p<0.05 can be considered statistically significant difference between groups.3.3 Univariate and multivariate logistic regression analyses were used to establish the odds ratios(ORs)for DR.Part Two In vitro study1.To verify the effects of glucose on human retina microvascular pericytes(HRMVPC),we cultured the HRMVPC-immortalized cell line in vitro and cells were treated with different concentrations of glucose(0,10,20,30 mM)for 24 h.1.1 Western blot was used to observe the expression of Bcl-2 family proteins,including Bcl-2 and Bax.1.2 30 mM glucose(high glucose)was selected for further experiments.Annexin V-FITC/PI flow cytometry and TUNEL dye were used to detect the apoptosis rate.1.3 MitoSOXTM Red dye was used to observe the accumulation of reactive oxygen species(ROS)in mitochondria.1.4 JC-1 dye was used to detect the level of mitochondrial membrane potential(MMP).1.5 Malondialdehyde(MDA)detection kit was used to examine lipid peroxidation damage.1.6 Chemiluminescence was used to detect the level of ATP synthesis.1.7 Western blot was used to observe the expression of the key proteins of mitochondrial apoptosis,including cleaved-caspase 9,cleaved-caspase 3,and the pro-apoptotic proteins P53 and PDCD 5.2.To explore the effects of TSH on glucose-induced pericytes apoptosis,cells then were treated with different concentrations(0,1,2,4 ?M)of bovine TSH(bovine TSH,bTSH)on basis of high glucose for 24 h,the above detection methods were used again to examine the effects of bTSH on the mitochondrial apoptosis pathway induced by high glucose treatment.3.TSH needs to bind to its specific receptors on the surface of the cell membrane to exert its biological effects.To verify whether the pericytes express TSH receptor(TSH-receptor,TSHR),we used the human thyroid follicular epithelial cell line(Nthy-ori 3-1)as a positive control,western blot was used to detect the expression of TSHR from protein level,immunization fluorescence was used to detect its location in the cells,PCR was used to detect its expression from the mRNA transcription level.4.To further confirm whether TSHR expressed by pericytes is functional,cells were given endogenous phosphodiesterase inhibitor isobutylmethylxanthine(IBMX)for pretreatment for 30 min and then were treated with bTSH(2 ?M)or adenylate cyclase activator Forskolin(50 ?M)for 1 h,enzyme-linked immunosorbent assay was used to detect cAMP levels in cells.Chinese hamster ovarian cancer cell line(CHO)was used as a negative control.5.After transfecting cells with TSHR small interfering RNA(si-RNA),western blot was used to detect the expression of TSHR to determine the transfection efficiency.At the same time,the expression of mitochondrial apoptosis-related proteins Bcl-2,Bax,cleaved-caspase 9,cleaved-caspase 3,and pro-apoptotic proteins P53 and PDCD 5 were detected to determine whether TSHR participated in the pro-apoptotic process of TSH.Results:1.Serum TSH level is an independent risk factor for DR:1.1 Compared with the non-DR group,the DR group has a higher proportion of female patients(38.36%vs.31.12%,p=0.02),older age(61 vs.59 years old,p?0.001),longer duration of diabetes(16 vs.10 years,p<0.001),and higher systolic blood pressure(134 mmHg vs.131 mmHg,p?0.001).In addition,we observed the duration of diabetes in the DR group(61 years vs.59 years,p=0.001),systolic blood pressure(134 mmHg vs.131 mmHg,p=0.001),HbAlc levels(8.90%vs.8.60%,p=0.032)and TSH levels(2.54 IU/L vs.1.47 IU/L,p<0.001)were significantly higher than those in the non-DR group.There were no significant differences in BMI,diastolic blood pressure,serum lipid levels,smoking and drinking history between the two groups(p?0.05).1.2 There are significant differences in the prevalence of DR between stratified serum TSH levels(8.3%,25.5%,64.4%and 95.8%,p<0.001),suggesting that TSH levels may be related to DR.1.3 After adjusting for the influence of confounding factors such as age,gender,duration of diabetes,blood pressure,and HbAlc,the level of serum TSH was still positively correlated with the prevalence of DR,OR=2,294,95%Cl[1.925-2.733],p<0.001.1.4 There was no significant difference in the severity of DR between stratified serum TSH levels(0%,5.1%,8.8%and 4.3%,p=0.556),and there was no correlation between them.2.Glucose could induce cell apoptosis:The expression of Bcl-2 down-regulated and expression of Bax was up-regulated after cells were treated with glucose,and the Bax/Bcl-2 ratio increased(p<0.05)in a dose-dependent manner.At the same time,Annexin V-FITC/PI flow cytometry and TUNEL dye showed that the apoptosis rate of the high glucose(30 mM)group was increased.The above results confirmed that glucose could promote the occurrence of apoptosis.3.High glucose activated mitochondrial apoptosis pathway:High glucose treatment led to increased ROS accumulation in mitochondria and decreased MMP(p<0.05).At the same time,MDA levels were increased,ATP synthesis was decreased(p<0.05).It was confirmed that high glucose could cause structural and functional damage to mitochondria.Meanwhile,high glucose up-regulated the expressions of mitochondrial apoptosis-related proteins cleaved-caspase 9,cleared-caspase 3,and pro-apoptotic proteins P53 and PDCD 5,(p<0.05).4.bTSH exacerbated high glucose-induced apoptosis by activating mitochondrial apoptosis pathway:After cells were treated with bTSH on the basis of high glucose,Bcl-2 expression was down-regulated and Bax expression was up-regulated compared with high glucose group,the Bax/Bcl-2 ratio was further increased(p<0.05),and the apoptosis rate was increased(p<0.05).Meanwhile,bTSH could further aggravate mitochondrial structural and functional damage,and up-regulate the expression level of mitochondrial apoptosis-related proteins(p<0.05).This indicated that bTSH could further aggravate mitochondrial apoptosis induced by high glucose.5.Human retinal micro vascular pericytes express biologically active TSHR:Western blot and PCR confirmed the stable expression of TSHR in pericytes from protein and mRNA transcription levels,respectively.In addition,compared with control group,both Forskolin and bTSH could significantly increase the level of cAMP in pericytes(p<0.05).In negative control cells,after Forskolin stimulation,cAMP levels were increased(p<0.05),while levels of cAMP had no significant changes compared with its own control group after the treatment of bTSH(p? 0.05).These results clarified that human PCs presented functional TSHR.6.TSHR is involved in regulating the pro-apoptotic process of TSH:After transfecting si-RNA to down-regulate the expression of TSHR in pericytes,cells can resist the pro-apoptotic effect of bTSH.From this we concluded that TSHR played an indispensable role in TSH-induced PCs mitochondrial apoptosis,which meant TSH's pro-apoptotic effect on PC is TSHR-dependent.Conclusion:1.The duration of T2DM,level of HbAlc and serum TSH level are the independent risk factors for DR.2.Serum TSH levels in T2DM patients are positively correlated with the prevalence of DR,but there is no obvious correlation with the prevalence of PDR,which means TSH levels were not correlated with the severity of DR.3.High glucose could induce mitochondrial apoptosis in human PCs.4.TSH could worsen high glucose-induced PCs apoptosis.5.Human retinal micro vascular PCs can stably present functional TSHR.TSH could aggravate high glucose induced endogenous apoptosis of PCs through TSHR-dependent mitochondrial apoptosis pathway. |
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