The Regulation Of Visfatin Expression In Neonatal Rat Heart Cells Under High Glucose

Hyperglycemia is the main signs and pathophysiological basis ofdiabetes.Diabetic cardiomyopathy is one of the cardiovascular complicationsof diabetes.Diabetes patients with cardiomyopathy myocardial injury mainlycomes from myocardial cells and myocardial mesenchymal cells twoaspects.In the past,there had been a lot of research on myocardial cell injury.Inrecent years, more and more study found that the interstitial fibrosis ofmyocardial also play an essential role in the occurrence and development ofdiabetic cardiomyopathy.The remodeling and fibrosis of myocardial interstitialare characteristic pathological manifestations of diabetic cardiomyopathy.Modern studies have shown that visceral adipose tissue, as the most importantendocrine organ, could secrete various cytokines such as C-reactive protein(C-of reactive protein, CRP), tumor necrosis factor-alpha (tumor necrosisfactor-alpha, TNF-alpha), interleukin-6(interleukin-6, IL-6),adiponectin,resistin, and leptin.These cytokines are closely related to the severity ofobesity-related diseases such as atherosclerosis, coronary heart disease,hypertension and diabetes.Visfatin is an adipokine identified in2005,because it was originally foundin the visceral fat cells, so named visfatin. Subsequent studies found that the5'noncoding sequences of visfatin is the same as PBEF. Visfatin has the samestructure as nicotinamide phosphoribosyltransferase (Nampt), the limitingenzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis.In additionto being produced in human leukocytes and adipose tissue,visfatin is alsoexpressed in hepatocytes,muscles and kidney of human and animal.Visfatinplays different biological activity effections in different tissues. Visfatininfluences the process and development of cardiovascular disease, andendothelial dysfunction, insulin resistance, diabetes and so on. Visfatin has the effect of insulin, promoting adipose cell differentiation, inhibiting cellapoptosis, promoting cells mature, proliferation and promoting inflammationfunctions. Studies have found that visfatin, as a growth stimulating factor,would be like fat even element, leptin that can be generated by heart itself,feedback effects on the heart, and play the insulin effect of inflammatorycytokines, proliferation, and anti-apoptotic role. However, few studies are onthe visfatin expression of heart cells.Diabetes cardiomyopathy is one of themain complications of diabetes cardiovascular.So far there has not been thereport about the synthesis and expression of visfatin on heart cells underhigh-sugar environment at home and abroad.In this study, we expose Sprague-Dawley (SD) neonatal rat myocardialcells and cardiac fibroblasts to the high concentration of glucose to explore theexpression of visfatin in myocardial cells and cardiac fibroblastsand.At thesame time,we discuss regulation and impaction of visfatin on myocardialfibrosis.Provide an important theoretical basis and guidance for the clinicaltreatment of diabetes, decrease the myocardial damage, and provide newtherapeutic targets for diabetic cardiomyopathy.Part1The regulation of visfatin expression about the neonatal rat myocardialcells under high glucose by MAPK signal transduction pathwayObjective: Exposing SD neonatal rat myocardial cells to differentconcentrations and different times,observing the expression of visfatin, toexplore the regulation of visfatin expression unde high glucose by differentMAPK signal transduction pathways.Methods: Extraction, cultured neonatal SD rat cardiomyocytes,(1)exposing myocardial cells to different concentrations and detection the visfatinexpression:①c ontrol group(normal glucose5.5mmol/L),②10mmol/L glucosegroup,③30mmol/L glucose group,④50mmol/L glucose group,⑤hypertoniccontrol group(5.5mmol/L glucose+44.5mmol/Lmannitol).(2)Exposingmyocardial cells to the same concentration of glucose, intervention at differenttimes and detection visfatin expression: the control group. Cardiomyocytesexposed to the glucose concentration of30mmo1/L.We detected at 6h,12h,24h and48h.⑶Interventing with different signal transduction pathwayinhibitors and detection the changes of visfatin expression:①controlgroup(normal glucose5.5mmol/L),②h igh glucose(30mmol/l),③high glucoseand SP600125,④high glucose and PD098059,⑤high glucose and SB203580.The activity of myocardial cells is measured by MTT assay. RT-PCR is used todetect the expression of myocardial visfatin mRNA and Western blot is usedto detect visfatin protein.Results:1The affect of different concentrations of glucose on the expressions ofVisfatin-mRNA/protein in cardiac myocytes.⑴MTT results showed:differentconcentrations (5.5mmol/L,10mmol/L and30mmol/L, and50mmol/L)glucose-stimulated48hours,with the increasing glucose concentration, theabsorbance values is in a dose group dependent increase and in50mmol/L topeak.⑵At different concentrations(10mmol/L,30mmol/L,50mmol/L)glucose-stimulated, In addition to hypertonic group, Visfatin-mRNAexpression than the control group had increased, and there was statisticallysignificant differences(P <0.01). Stimulating with different concentration (10mmol/L,30mmol/L) glucose,the visfatin-mRNA expression of myocardialcell increased with a dose dependentment, while the concentration of50mmol/L group expression of Visfatin in-mRNA compared with30mmol/Lgroup reduced, but there was no significant difference between the two groups.⑶At different concentrations(10mmol/L,30mmol/L,50mmol/L)glucose-stimulated, Visfatin protein expression than the control group hadincreased, and there was statistically significant differences(P <0.01). Proteinexpression in30mmol/L group is the most. The concentration of50mmol/Lgroup expression of Visfatin-Protein compared with30mmol/L group reduced,there was significant difference between the two groups(P <0.05). Theexpression of Visfatin in hypertonic group is lower than5.5mmol/L group,and there was statistically significant difference (P <0.05). These resultssuggest that: high glucose cause myocardial cells express Visfatinincrease.Within a certain concentration the increase expression was in concentration-dependent,and this role has nothing to do with hypertonicenvironment caused by high glucose.2High glucose (30mmol/L) stimulates the cardiac cells at different timesand oberve the expression of Visfatin-mRNA/protein.⑴MTT results show:30mmol/L glucose at different times (12,24and48hours) stimulation, thereis a significant increase in each group absorbance values than in the controlgroup.There is a significant difference(P<0.01). Within24hours,increase withstimulation time, the absorbance values showed a time dependent increase.Thepeak is at24hours,and began to decrease after48hours.Compared to24hgroup,there is a the significant difference (P <0.05).⑵30mmol/L glucose atdifferent times (6,12,24and48hours) stimulation, compared with the controlgroup,there is a significant increase in Visfatin mRNA/protein expression ofeach group, there is a significant difference(P<0.05); within24hours, increasewith stimulation time, the expression of Visfatin mRNA/protein showed a timedependent increase.The peak is at24hours,and began to decrease after48hours.Compared to24h group,there is no significant difference. These resultssuggest that: within a certain time frame, with the extension of time underhigh concentrations of glucose, the visfatin expression in cardiac myocytesincreased.3The regulation of visfatin expression unde high glucose by MAPK signaltransduction pathway: after the intervention of the different MAPK signaltransduction pathway inhibitors, compared to the control group (low glucose5.5mmol/L),the expression of Visfatin-mRNA/protein in high glucose30mmol/L group, high glucose30mmol/L+PD98059, group, high glucose30mmol/L+SP600125group,and high glucose30mmol/L+SB203580groupincreased.There was statistically significant differences(P <0.01). However,Visfatin mRNA/protein expression in high glucose30mmol/L+SB203580group was significantly lower than high glucose30mmol/L group. There wassignificant difference(P <0.05). Compared high glucose30mmol/L+PD98059group, high glucose30mmol/L+SP600125group with the highglucose30mmol/L group,there was no statistically significant differences. These results suggest that: SB203580inhibits the Visfatin expression ofmyocardial cells induced by high glucose.Conclusion: High glucose stimulates the synthesis of Visfatin inmyocardial cells, and within a certain time and concentration, the increaseexpression was in concentration-time-dependent. P38MAPK signaltransduction pathways involved in mediating the Visfatin expression ofmyocardial cell under the high-sugar environment.Part2The regulation of visfatin and collagen Ⅰexpression about the neonatalrat cardiac fibroblasts under high glucose by Rho/ROCK signal transductionpathwaysObjective: Exposing SD neonatal rat cardiac fibroblasts to differentconcentrations and different time and observing the expression of visfatin andcollagenⅠ, to explore the regulation of visfatin and collagen Ⅰexpressionabout the neonatal rat cardiac fibroblasts under high glucose by RhoA/Rhokinase signal transduction pathways.Methods: Extraction, cultured neonatal SD rat cardiac fibroblasts,(1)exposing cardiac fibroblasts to different concentrations and detection theexpression of visfatin and precollagenⅠ:①c ontrol group(normal glucose5.5mmol/L),②10mmol/L glucose group,③30mmol/L glucose group,④50mmol/L glucose group,⑤h ypertonic control group(5.5mmol/L glucose+44.5mmol/Lmannitol).(2)Exposing cardiac fibroblasts to the sameconcentration of glucose, intervention at different times and detection theexpression of visfatin and precollagenⅠ.Cardiac fibroblasts exposed to theglucose concentration of30mmo1/L.We detected at6h,12h,24h and48h.(3)The regulation of expression of visfatin, precollagen Ⅰa ndROCK1under highglucose by Rho/ROCK signal transduction pathways.①control group(normalglucose5.5mmol/L),②high glucose(30mmol/l),③l ow glucose (5.5mmol/L)and Y27632,④h igh glucose andY27632. The activity of cardia fibroblasts ismeasured by MTT assay. Real time-PCR is used to detect the expression ofvisfatin, precollagen ⅠmRNA and Western blot is used to detect protein ofvisfatin, precollagenⅠ andROCK1. Results:1The affect of different concentrations of glucose on the expressions ofVisfatin, precollagenⅠ-mRNA/protein in cardiac fibroblasts.⑴MTT resultsshowed:different concentrations (5.5mmol/L,10mmol/L and30mmol/L, and50mmol/L) glucose-stimulated48hours,with the increasing glucoseconcentration, the absorbance values is in a dose group dependent increaseand in30mmol/L to peak. The absorbance values of50mmol/L groupcompared with30mmol/L group reduced, but there was no significantdifference between the two groups.⑵At different concentrations(10mmol/L,30mmol/L,50mmol/L) glucose-stimulated, In addition to hypertonic group,precollagenⅠ-mRNA/protein expression than the control group had increasedwith a dose dependentment, and there was statistically significant differences(P<0.01). While the concentration of50mmol/L group expression ofprecollagenⅠ-mRNA/protein is most. These results suggest that: high sugarcause cardiac fibroblasts express precollagenⅠincrease with aconcentration-dependent,and this role has nothing to do with hypertonicenvironment caused by high glucose.⑶At different concentrations(10mmol/L,30mmol/L,50mmol/L) glucose-stimulated, Visfatin-mRNA/proteinexpression than the control group had increased, and there was statisticallysignificant differences(P <0.01). Visfatin expression in30mmol/L group isthe most. The concentration of50mmol/L group expression of Visfatincompared with30mmol/L group reduced, there was no significant differencebetween the two groups(P>0.05). The expression of Visfatin in hypertonicgroup is lower than5.5mmol/L group, and there was statistically significantdifference (P>0.05). These results suggest that: high sugar cause cardiacfibroblasts express Visfatin increase.Within a certain concentration theincrease expression was in concentration-dependent,and this role has nothingto do with hypertonic environment caused by high glucose.2High glucose (30mmol/L) stimulates the cardiac fibroblasts at differenttimes and oberve the expression of Visfatin, precollagenⅠ-mRNA/protein.⑴MTT results show:30mmol/L glucose at different times (6,12,24and48 hours) stimulation, there is a significant increase in each group absorbancevalues than in the control group.There is a significant difference(P <0.05).Within48hours,increase with stimulation time, the absorbance values showeda time dependent increase.The peak is at48hours.⑵30mmol/L glucose atdifferent times (6,12,24and48hours) stimulation, compared with the controlgroup,there is a significant increase in precollagenⅠ-mRNA/proteinexpression of each group, there is a significant difference(P<0.01); within24hours, increase with stimulation time, the precollagen-mRNA/protein showeda time dependent increase.The peak is at24hours,and began to decrease after48hours.Compared to24h group,there is no significant difference(P>0.05).These results suggest that: within a certain time frame, with the extension oftime under high concentrations of glucose, the precollagenⅠ expression incardiac fibroblasts increased.⑶30mmol/Lglucose at different times (12,24and48hours) stimulation, compared with the control group,there is asignificant increase in Visfatin-mRNA/protein expression of each group, thereis a significant difference(P<0.01); within24hours, increase with stimulationtime, the Visfatin-protein showed a time dependent increase.The peak is at24hours,and began to decrease after48hours.Compared to24h group,there is nosignificant difference(P>0.05). These results suggest that: within a certain timeframe, with the extension of time under high concentrations of glucose, theVisfatin expression in cardiac fibroblasts increased.3The regulation of visfatin,precollagenⅠ a ndROCK1under high glucose(30mmol/L)by Rho/ROCK signal transduction pathway:⑴compared to thecontrol group (low glucose5.5mmol/L),the expression of precollag-enⅠ-mRNA/protein in high glucose30mmol/L group and high glucose30mmol/L+Y27632increased.There was statistically significant differences(P<0.01). Moreover,precollagenⅠ-mRNA/protein expression in high glucose30mmol/L+Y27632group was significantly lower than high glucose30mmol/L group. There was significant difference(P <0.05). Compared withthe control group, the expression of precollagenⅠ-mRNA/protein in lowglucose5.5mmol/L+Y27632was no statistically significant differences (P>0.05). These results suggest that: Y27632inhibits the precollagenⅠexpression of cardiac fibroblasts induced by high glucose.⑵compared to thecontrol group (glucose5.5mmol/L),the expression of visfatin-mRNA in highglucose30mmol/L group increased.There was statistically significantdifferences(P<0.01). Moreover, visfatin-mRNA expression in high glucose30mmol/L+Y27632group was significantly lower than high glucose30mmol/L group. There was significant difference(P <0.05). Compared withthe control group, the expression of visfatin-mRNA in high glucose30mmol/L+Y27632and low glucose5.5mmol/L+Y27632was nostatistically significant differences(P>0.05). Compared to the control group(glucose5.5mmol/L),the expression of visfatin protein in high glucose30mmol/L group and high glucose30mmol/L+Y27632increased.There wasstatistically significant differences(P <0.01). Moreover, visfatin proteinexpression in high glucose30mmol/L+Y27632group was significantly lowerthan high glucose30mmol/L group. There was significant difference(P <0.05).Compared with the control group, the expression of visfatin protein in lowglucose5.5mmol/L+Y27632was no statistically significant differences(P>0.05). These results suggest that: Y27632inhibits the visfatin expressionof cardiac fibroblasts induced by high glucose.⑶compared to the controlgroup (low glucose5.5mmol/L),the expression of ROCK1protein in highglucose30mmol/L group increased.There was statistically significantdifferences(P <0.01).,the expression of ROCK1protein expression in highglucose30mmol/L+Y27632group was significantly lower than high glucose30mmol/L group. There was significant difference(P <0.05). Compared withthe control group, the expression of ROCK1protein in low glucose5.5mmol/L+Y27632and high glucose30mmol/L+Y27632group was nostatistically significant differences (P>0.05). These results suggest that:Y27632inhibits the ROCK1expression of cardiac fibroblasts induced by highglucose.Conclusion: High glucose stimulates the synthesis of Visfatin andcollagen Ⅰ in cardiac fibroblasts, and within a certain time and concentration, the increase expression was in concentration-time-dependent. Rho/ROCKsignal transduction pathways involved in mediating the Visfatin andcollagen Ⅰ expression of cardiac fibroblasts under the high-sugar environment.Part3The influence of Visfatin to the collagen synthesis of neonatal ratcardiac fibroblast under the stimulation of high glucoseObjective: Incubating SD neonatal rat cardiac fibroblasts in vitro andblocking the role of Visfatin,observe the impact on synthesis of precollagenⅠand precollagen Ⅲof neonatal rat cardiac fibroblast cell. In order to explorethe mechanism of the Visfatin in myocardial fibrosis of diabetescardiomyopathy.Methods: Extraction, cultured neonatal SD rat cardiac fibroblasts,①control group(normal glucose5.5mmol/L),②high glucose(30mmol/l),③lowglucose (5.5mmol/L) and FK866,④h igh glucose andFK866. Real time-PCRis used to detect the expression of precollagen Ⅰ andprecollagen ⅢmRNA ofneonatal rat cardiac fibroblast and Western blot is used to detect protein ofcollagen Ⅰ and collagen Ⅲ.Results:1The influence of precollagenⅠ-mRNA/protein under high glucose(30mmol/L)by FK866: compared to the control group,the expression ofprecollagenⅠ-mRNA in high glucose30mmol/L group and high glucose30mmol/L+FK866group was increased.There was statistically significantdifferences(P <0.01). Compared with the control group, the expression ofprecollagen Ⅰ mRNAin low glucose5.5mmol/L+FK866group was nostatistically significant differences(P>0.05). Moreover,precollagn ⅠmRNAexpression in high glucose30mmol/L+FK866group was significantly lowerthan high glucose30mmol/L group. There was significant difference(P <0.05).compared to the control group,the expression of precollagenⅠprotein in highglucose30mmol/L group was increased.There was statistically significantdifferences(P <0.01). Compared with the control group, the expression ofprecollagenⅠprotein in low glucose5.5mmol/L+FK866group and highglucose30mmol/L+FK866group was no statistically significant differences (P>0.05). Moreover,precollagenⅠ protein expression in high glucose30mmol/L+FK866group was significantly lower than high glucose30mmol/L group. There was significant difference(P <0.05). These resultssuggest that: FK866inhibits the precollagenⅠ expression of cardiacfibroblasts induced by high glucose.2The influence of precollagenⅢ-mRNA/protein under high glucose(30mmol/L)by FK866:compared to the control group (low glucose5.5mmol/L),the expression of precollagenⅢ-mRNA/protein in high glucose30mmol/L group and high glucose30mmol/L+FK866group wasincreased.There was statistically significant differences(P <0.01). Comparedwith the control group, the expression of precollagenⅢ-mRNA/protein in lowglucose5.5mmol/L+FK866group awas no statistically significantdifferences(P>0.05). Moreover,precollagnⅢ-mRNA/protein expression inhigh glucose30mmol/L+FK866group was significantly lower than highglucose30mmol/L group. There was significant difference(P <0.05). Theseresults suggest that: FK866inhibits the precollagen Ⅲexpression of cardiacfibroblasts induced by high glucose.Conclusion: The Visfatin involved in the process of cardiac fibroblastand collagen synthesis of of cardiac fibroblast induced by high glucose.Part4The effect of rosiglitazone on the Visfatin expression and collagensynthesis of neonatal rat cardiac fibroblast on high glucose-inducedObjective: Incubating SD neonatal rat cardiac fibroblasts in vitro andafter the intervention of rosiglitazone,observe the impact oncollagen Ⅰsynthesis and the Visfatin expression of neonatal rat cardiacfibroblast cell. In order to explore the mechanism of the rosiglitazone inmyocardial fibrosis of diabetes cardiomyopathy.Methods: Extraction, cultured neonatal SD rat cardiac fibroblasts,①control group(normal glucose5.5mmol/L),②high glucose(30mmol/l),③lowglucose (5.5mmol/L) and rosiglitazone,④high glucose(30mmol/l) androsiglitazone.Real time-PCR is used to detect the expression of precollagenⅠand Visfatin mRNA of neonatal rat cardiac fibroblast and Western blot is used to detect protein of precollagenⅠ and Visfatin.Results:1The influence of precollagenⅠ-mRNA/protein under high glucose (30mmol/L)by rosiglitazone:compared to the control group,the expression ofprecollagenⅠ-mRNA/protein in high glucose30mmol/L group and highglucose30mmol/L+rosiglitazone group was increased.There was statisticallysignificant differences(P <0.01). Compared with the control group, theexpression of precollagenⅠ-mRNA/protein in low glucose5.5mmol/L+rosiglitazone group was no statistically significant differences (P>0.05).Moreover,precollagenⅠ-mRNA/protein expression in high glucose30mmol/L+rosiglitazone group was significantly lower than high glucose30mmol/Lgroup. There was significant difference(P <0.05). These results suggest that:rosiglitazone inhibits the precollagenⅠ expression of cardiac fibroblastsinduced by high glucose.2The influence of Visfatin-mRNA/protein under high glucose (30mmol/L)byrosiglitazone:compared to the control group,the expression of Visfatin-mRNA/protein in high glucose30mmol/L group and high glucose30mmol/L+rosiglitazone group was increased.There was statistically significantdifferences(P <0.05). Compared with the control group, the expression ofVisfatin-mRNA/protein in low glucose5.5mmol/L+rosiglitazone group wasno statistically significant differences(P>0.05). Moreover, Visfatin-mRNA/protein expression in high glucose30mmol/L+rosiglitazone group wassignificantly lower than high glucose30mmol/L group. There was significantdifference(P <0.05). These results suggest that: rosiglitazone inhibits theVisfatin expression of cardiac fibroblasts induced by high glucose.Conclusion: Rosiglitazone inhibit the expression of collagen secretionand Visfatin with cardiac fibroblast in the environment of hyperglycemia,maybe plays a role in the process of myocardial fibrosis.