The Intervention Mechanisms Of ?-hydroxybutyrate On Diabetic Micro-cardiovascular Disease By Antagonizing Nitration

Objective: Diabetes mellitus(Diabetes mellitus,DM)is a chronic metabolic disease caused by hyperglycemia,the number of patients is expected to rise to 590 million by 2035 all over the world.The pathological of micro-cardiovascular is a characteristic feature in heart of diabetic,the typical pathological changes is the proliferation of vascular basement membrane and lead to microcirculatory disturbances.Microvascular endothelial dysfunction could induced the change of major component of vascular basement membrane in diabetic,the pathological base of which is accumulation of collagen ?,the persistent nitration caused by oxidative stress is its core pathogenesis.The production of collagen ? which is the main component of vascular basement membrane,was regulated by the pathway of TGF-?1.The Signaling of TGF-?1 could activate the gene transcription of collagen ? by Smad3.Study found that Smad3 was knocked out,the alpha 1 chain of collagen ? was not expressed.By far,the underlying mechanism of the proliferation of vascular basement membrane was not clarified,and there were no special effects of clinical treatments yet.Further research on the mechanism of collagen ? accumulation,to provide new thinking of the prevention and treatment of diabetic micro-cardiovascular disease.Bioactive Small Molecules ketone bodies was composed by ?-hydroxybutyrate(seventy percent),acetoacetate(thirty percent)and acetone(trace amounts),which has many protection functions in cardiovascular system.It is reported that the major component of ketone bodies,?-hydroxybutyrate has good effect of inhibiting the cell injury induced by oxidative stress,especially in cardiovascular system.Srudies founded that different from conventional antioxidants,?-hydroxybutyrate could take part in regulating gene expression.Moreover,?-hydroxybutyrate also could be used as endogenous and specific inhibitors of class?HDAC,selectively increasethe level of histone acetylation in the promoter region of the protective gene,and play a significant role against the process of oxidative stress.In this study,the human cardiac microvascular endothelial cells were ussed.Firstly,in order to determine the optimal concentration of high glucose condition just like the state in diabetes,cells were exposed to different concentrations of high glucose;then HCMEC cells were treamented by different concentrations of ?-hydroxybutyrate at the same concentration of glucose medium and action time;RT-PCR was performed to assay the mRNA expressions of IV?1,iNOS and MMP-9.the protein level of collagen ? in culture media was measured in HCMEC cells using enzyme linked linked immunosorbent assay(ELISA)detection kits.Western blot was performed to assay the protein level of P300,Smad3,HDAC3 and nitration;CoImmunoprecipitation(Co-IP)assay was used to detect respectively the binding effects of Smad3 with P300 and HDAC3.The cell immunofluorescent staining application was performed to detect the content of NT in HCMEC cells.All in all,this study aimed to explore the major molecular mechanism of?-hydroxybutyrate in regulating collagen ? metabolism balance by antagonizing nitration,to provide experimental evidence for the therapy.Methods:1 Cell culture: The human cardiac microvascular endothelial cells(HCMEC)was purchased from the United States model bacteria collection center(ATCC).HCMEC cells were propagated in low sugar Endothelial Cell Medium(ECM)containing 5% fetal bovine serum(FBS),cells were inoculated in 250 mL flasks and passaged with 0.25% trypsin.Then the cells were maintained at 37°C in a humidified incubator with 5% CO2.Cells treated with ?-hydroxybutyric acid,SIN-1 and FeTPPs,the cells of SIN-1 group were cultured with low sugar medium plus SIN-1,while the cells of ?-hydroxybutyric acid and FeTPPS groups were cultured with 30 mM high D-glucose ECM plus ?-hydroxybutyrate and FeTPPs respectively.2 Experimental design and cell grouping2.1 To determine the effect of high D-glucose at various concentrationson HCMEC cells,the mRNA expressions of ??1,MMP-9 and i NOS.HCMEC cells were exposed to different concentrations of D-glucose for24-hour,According to different concentrations of sugar,the experiment was divided into five groups: Con(control)group(with ECM containing 5.5 mM D-glucose),20 mM group(with ECM containing 20 mM D-glucose),25 mM group(with ECM containing 25 mM D-glucose),30 mM group(with ECM containing 30 mM D-glucose)and 35 mM group(with ECM containing 35 mM D-glucose).2.2 To investigate the different concentrations effects of?-hydroxybutyrate on HCMEC cells,the mRNA expression of ? ?1,MMP-9 and i NOS.According to different concentrations of ?-hydroxybutyrate,the experiment was divided into five groups: Con(control)group(cells were incubated in 5.5 mM D-glucose ECM),HG group(cells were incubated in 30 mM D-glucose ECM),?2 group(cells were incubated in 30 mM D-glucose ECM plus 0.02 mM ?-hydroxybutyrate),?4 group(cells were incubated in 30 mM D-glucose ECM plus 0.04 mM ?-hydroxybutyrate),?6 group(cells were incubated in 30 mM D-glucose ECM plus 0.06 mM ?-hydroxybutyrate).3 The changes of ? ?1 genes transcriptional level were detected in HCMEC cells.Total RNA was isolated using a one-step Trizol reagent in HCMEC cells.RT-PCR analysis for the expression of mRNA for ??1 genes with ?-actin as a control reference was carried out.4 The protein level of collagen ? in culture media was measured by ELSIA;The Western blot assay was performed to confirm the protein level of P300,Smad3,HDAC3 and nitration;Co-IP assay was used to detect the binding activity of Smad3 with P300 and HDAC3 in HCMEC cells.The experiment was divided into five groups: Con(control)group(cells were incubated in 5.5 mM D-glucose ECM),HG group(cells were incubated in 30 mM D-glucose ECM),SIN-1 group(cells were incubated in 5.5 mM D-glucose ECM plus 5 mM SIN-1),?-HB group(cells were incubated in 30 mM D-glucose ECM plus 0.04 mM ?-HB)and FeTPPs group(cells were incubated in 30 mM D-glucose ECM plus 5 mM FeTPPs).5 The cell immunofluorescent staining application was performed to detect the content of NT in HCMEC cells.6 Statistical analysisAll the experiments were repeated three times,data are expressed as mean ± SD,SPSS16.0 software was used for statistical analysis,and all the datas were deal with One-Way-ANOVA,differences were considered significant at a value of P<0.05.Results:1 RT-PCR was performed to assay the mRNA expressions of ? ?1,MMP-9 and iNOS in HCMEC cells stimulated with different concentrations of D-glucose for 24-hour.The ??1 mRNA relative expression level in the 20 mM(2.56 ± 0.06,P<0.05),25 mM(2.43 ± 0.18,P<0.01),30 mM(4.97 ± 0.46,P<0.01)and 35mM(2.41 ± 0.17,P<0.01)groups was significantly higher than those in the Con group.The MMP-9 mRNA relative expression level in the 20 mM(0.16 ±0.03,P<0.05),25 mM(0.09 ± 0.04,P<0.01),30 mM(0.02 ± 0.01,P<0.01)and 35 mM(0.05 ± 0.02,P<0.05)groups was significantly higher than those in the Con group.The iNOS mRNA relative expression level in the 20 mM(1.30 ± 0.19,P<0.05),25 mM(2.12 ± 0.03,P<0.01),30 mM(3.05 ± 0.05,P<0.01)and 35 mM(2.07 ± 0.02,P<0.05)groups was significantly higher than those in the Con group.2 RT-PCR was performed to assay the mRNA expressions of ? ?1,MMP-9 and iNOS in HCMEC cells stimulated with different concentrations of ?-hydroxybutyrate.The ??1 mRNA relative expression level in the ?2(0.27 ± 0.02,P<0.05),?4(0.15 ± 0.01,P<0.01)and ?6(0.32 ± 0.02,P<0.01)groups was significantly lower than those in the HG group.The MMP-9 mRNA relative expression level in the ?2(1.10 ± 0.01,P<0.05),?4(1.73 ± 0.03,P<0.01)and ?6(0.38 ± 0.02,P<0.01)groups was significantly higher than those inthe HG group.The iNOS mRNA relative expression level in the ?2(0.56 ±0.03,P<0.05),?4(0.15 ± 0.01,P<0.01)and ?6(0.32 ± 0.02,P<0.01)groups was significantly lower than those in the HG group.3 RT-PCR was performed to assay the mRNA expressions of ?1(?)in HCMEC cells.The ?1(?)mRNA relative expression level in the HG(2.17 ± 0.11,P<0.05)and SIN-1(1.94 ± 0.08,P<0.05)groups was significantly higher than groups in contrast with Con group;?-HB(1.21 ± 0.10,P < 0.05)and FeTPPs(0.93 ± 0.08,P<0.01)groups was significantly lower than groups in contrast with HG group.4 ELISA assay was performed to assess the protein level of collagen ?in culture media in HCMEC cells.The protein level of collagen ? in culture media was significantly higher in HG(6.58 ±0.04 pg/mL)(P<0.05)and SIN-1(4.57 ± 0.07 pg/mL)(P<0.05)groups than the Con group.While that was no overt significance in the ?-HB(3.21±0.12 pg/mL)(P<0.01)and FeTPPs(3.33 ± 0.01 pg/mL)(P<0.01)groups compared to the Con group.5 Western blot assay was performed to evaluate the protein level of P300,Smad3,iNOS,HDAC3 and NT with different treaments in HCMEC cellsThe relative protein expression of P300 in the HG group(0.42 ± 0.02,P<0.01)and SIN-1 group(0.29 ± 0.01,P < 0.05)were significantly higher than those in the Con group(0.14 ± 0.01,P<0.05);compared with the HG group,the ?-HB group(0.17 ± 0.01,P<0.01)and the Fe TPPs group(0.15 ±0.01,P<0.05)were both decrease.The relative protein expression of Smad3 in the HG group(0.96 ± 0.01,P<0.05)and SIN-1 group(0.85 ± 0.03,P<0.05)were significantly higher than those in the Con group(0.30 ± 0.02,P<0.05);compared with the HG group,the ?-HB group(0.46 ± 0.02,P<0.01)and the FeTPPs group(0.82 ± 0.01,P<0.05)were both decrease.The relative protein expression of HDAC3 in the HG group(0.24 ± 0.02,P<0.01)and SIN-1 group(0.19 ± 0.01,P<0.05)were significantly higher than those in the Con group(0.13 ± 0.01,P<0.05);compared with the HG group,the ?-HBgroup(0.14± 0.01,P<0.01)and the FeTPPs group(0.17 ± 0.01,P<0.05)were both decrease.The relative protein expression of NT in the HG group(0.42 ± 0.02,P < 0.01)and SIN-1 group(0.39 ± 0.02,P < 0.05)were significantly higher than those in the Con group(0.11 ± 0.01,P < 0.05);compared with the HG group,the ?-HB group(0.17 ± 0.02,P<0.01)and the FeTPPs group(0.15 ± 0.01,P<0.01)were both decrease.6 Co-IP assay was performed to evaluate the effects of Smad3 with P300 and HDAC3 in different treatments of HCMEC cells6.1 To evaluate the effects of Smad3 with P300The cell protein lysates of the Con?HG?SIN-1??-HB and FeTPPs group were added to Smad3 antibody precipitation respectively,the analysis of Western blot results showed that the binding content of coimmunoprecipitated Smad3 and P300 in HCMEC cells of HG group(4.79 ±0.27,P < 0.01)were significantly higher than that in Control group(1.40±0.17);while he binding content of co-immunoprecipitated Smad3 and P300 in HCMEC cells of ?-HB(2.06 ± 0.31,P<0.01)and FeTPPs group(2.98 ± 0.03,P<0.05)were significantly decreased compared with the HG group.The cell protein lysates of the Con?HG?SIN-1??-HB and FeTPPs group were added to P300 antibody precipitation respectively,the analysis of Western blot results showed that the binding content of co-immunoprecipitated P300 and Smad3 in HCMEC cells of HG group(5.24± 0.18,P<0.01)were significantly higher than that in Control group(1.23 ±0.07);while he binding content of co-immunoprecipitated P300 and Smad3 in HCMEC cells of ?-HB(2.11 ± 0.26,P<0.01)and FeTPPs(3.46 ± 0.10,P<0.05)group were significantly decreased compared with the HG group.6.2 To evaluate the effects of Smad3 with HDAC3The cell protein lysates of the Con?HG?SIN-1??-HB and FeTPPs group were added to Smad3 antibody precipitation respectively,the analysis of Western blot results showed that the binding content of coimmunoprecipitated Smad3 and HDAC3 in HCMEC cells of HG group(3.47± 0.18,P<0.01)were significantly higher than that in Control group(1.21 ±0.11);while he binding content of co-immunoprecipitated Smad3 and HDAC3 in HCMEC cells of ?-HB(1.72 ± 0.17,P<0.01)and FeTPPs(2.39 ± 0.19,P<0.05)group were significantly decreased compared with the HG group.The cell protein lysates of the Con?HG?SIN-1??-HB and FeTPPs group were added to antibody precipitation respectively,the analysis of Western blot results showed that the binding content of co-immunoprecipitated HDAC3 and Smad3 in HCMEC cells of HG group(4.15 ± 0.25,P < 0.01)were significantly higher than that in Control group(1.28 ± 0.15);while he binding content of co-i mmunoprecipitated HDAC3 and Smad3 in HCMEC cells of?-HB(1.87 ± 0.10,P<0.01)and FeTPPs(2.03 ± 0.23,P<0.05)group were significantly decreased compared with the HG group.7 The cell immunofluorescent staining application was utilized to detect the level of NT in HCMEC cells.The fluorescence Intensity of NT in the HG and SIN-1 groups were significantly ehhanced compared with the Con group,especially in the HG group;while compared with the HG group,the fluorescence Intensity of NT in the ?-HB and FeTPPs groups were significantly weakened.Conclusion:1 ?-hydroxybutyrate can significantly inhibit the expression of ? ?1mRNA in HCMEC cells which were induced by high glucose(30 mM),It was0.04 mM ?-hydroxybutyrate that had the best inhibitory effect.2 ?-hydroxybutyrate can significantly inhibit the protein expression of Smad3 in HCMEC cells induced by high glucose(30 mM).3 ?-hydroxybutyrate could inhibite the interaction between Smad3 and P300 as well as between Smad3 and HDAC3 in high-glucose(30 mM)stimulated HCMEC cells.