Effect Of ALDH2 On High Glucose Induced Cardiac Fibroblasts Injury And The Changes Of MMP14/TIMP-4 Proteins

Objective: Our study was firstly to observe whether acetaldehyde dehydrogenase 2(ALDH2)was expressed in neonate rat cardiac fibroblasts(CFs),then to investigate the effect of ALDH2 on high glucose induced cardiac fibroblasts injury and the changes of matrix metalloproteinases(MMPs)proteins.Methods: The primary culture of cardiac fibroblasts was obtained from ventricles of 1~3 days old Sprague-Dawley(SD)suckling rats.Cardiac fibroblasts were randomly divided into seven groups: normal group,normal glucose+Alda-1 group,high glucose group,high glucose+Alda-1 group,high glucose+Alda-1+Daidzin group,high glucose+Daidzin group and hypertonic group.Cardiac fibroblasts were identified by immunofluorescence technique.For ensuring whether ALDH2 was expressed in cardiac fibroblasts,ALDH2 m RNA and protein expressions were determined by RT-PCR and Western blot.Cell proliferation was detected by MTT method in all groups.The level of reactive oxygen species was measured by DHE fluorescent probe.Apoptosis was observed via flow cytometer(Annexin V and propidiom iodide double staining method).The transformation of cardiac fibroblasts into myofibroblasts under high glucose and drug intervene was detected by immunofluorescence double staining method.The expressions of collagen type I and III at m RNA level were detected by real-time PCR.ALDH2,MMP-14 and TIMP-4 protein expressions in cardiac fibroblasts were tested by Western blot.Results: RT-PCR and Western blot results revealed ALDH2 was expressed in cardiac fibroblasts.Cell viability and proliferation in high glucose group,high glucose +Alda-1+Dainzin group and high glucose +Daidzin group were higher than those in normal group(P<0.01).When compared with high glucose group,the viability and proliferation in high glucose+Alda-1 were inhibited(P<0.01).The viability and proliferation were increased(P<0.05)in high glucose+Alda-1+Daidzin and high glucose +Daidzin groups compared with high glucose +Alda-1 group.The ROS releasing was higher significantly in high glucose group compared with normal group(P<0.01),and was lower in high glucose+Alda-1 group(P<0.01).When compared with high glucose+Alda-1 group,the ROS level was increased in high glucose+Alda-1+Daidzin group and high glucose +Daidzin group(P<0.01).The flow cytometry results indicated that the apoptosis rate in high glucose group,high glucose+Alda-1+Daidzin group and high glucose +Daidzin group were increased compared with normal group(P<0.01).The apoptosis rate in high glucose +Alda-1 group was decreased compared with high glucose group(P<0.01).When compared with high glucose +Alda-1 group,the apoptosis rates in high glucose +Alda-1+Daidzin group and high glucose+Daidzin group were increased significantly(P<0.01).The number of cardiac fibroblasts transformed into myofibroblasts was increased with the increasing time under high glucose stimulation.When compared with normal group,the m RNA expressions of Collagen I and Collagen III in high glucose group were increased(P<0.01,P<0.05).However,Collagen I and Collagen III expressions at m RNA level in high glucose+Alda-1 were decreased compared with high glucose group(P<0.01,P<0.05).When compared with high glucose+Alda-1,the m RNA expression of Collagen I in high glucose +Alda-1+Daidzin group was increased(P<0.01),the m RNA expression of Collagen I and Collagen III in high glucose +Alda-1+Daidzin and high glucose+Daidzin groups were increased(P<0.01,P<0.05).There was no significant difference of ALDH2,MMP-14 and TIMP-4 between normal group and normal glucose +Alda-1 group.When compared with normal group,the protein expressions of ALDH2 and MMP-14 in high glucose group,high glucose+Alda-1+Daidzin group and high glucose +Daidzin group were decreased(P<0.01,P<0.05),while TIMP-4 expression was increased(P<0.01).The protein expression of ALDH2 and MMP-14 in high glucose +Alda-1 were increased compared with high glucose group(P<0.01),while the expression of TIMP-4 was decreased(P<0.01).When compared with high glucose +Alda-1 group,the protein expression of ALDH2 and MMP-14 in high glucose +Alda-1+Daidzin group and high glucose +Daidzin group were decreased(P<0.01,P<0.05),while the expression of TIMP-4 in high glucose +Alda-1+Daidzin group was increased(P<0.01).Conclusions: ALDH2 was expressed in neonate rat cardiac fibroblasts.Activation of ALDH2 with Alda-1 may relieve the happening of fibrosis and apoptosis in high glucose induced cardiac fibroblasts injury model.The protective effect was related to the inhibition of oxidative damage,down-regulation of MMP-14 and up-regulation of TIMP-4 proteins.