| Objective Determine the effect of Danhong Injection(DHI)on the growth of myocardial cells(MCs)and investigate the protective effect of DHI on hypoxia/reoxygenation(H/R)'s MCs.Explore the protective mechanism of DHI onH/R's MCs based on the apoptosis,cAMP/PKA signaling pathway and NF-?B signaling pathway.Methods SD neonatal rats were used to obtain the MCs.After purified,MCs were randomly divided into normal,20%,10%,5%,2.5%,1.25%,0.63%,0.32%,0.16%,0.08%,0.04%DHI groups.After cultured in corresponding concentration of DHI for 24h,the cell activity was detect by MTT.MCs were randomly divided into normal,model,20%,10%,5%,2.5%,1.25%,0.63%,0.32%,0.16%,0.08%DHI groups.After cells were nearly in fusion state,corresponding concentration of DHI were added in the MCs,and made mould with H/R injury at the same time(except the normal group),then used MTT to detect the cell activity.MCs were randomly divided into 6 groups:normal,model,verapamil,20%,10%,5%DHI groups.Annexin V/PI double marking method was used to detect cell apoptosis on flow cytometer,after the 12 wells were covered with MCs,and then corresponding concentration of DHI and verapamil were added,made mould with H/R injury at the same time(except the normal group).MCs were randomly divided into 5 groups:normal,model,20%,10%,5%DHI groups.after the culture flasks were covered with MCs,corresponding concentration of DHI and verapamil were added,made mould with H/R injury at the same time(except the normal group),ELISA method,Western Blotting method and Fluorescence Quantitative RT-PCR method were used to detect the protein and mRNA expression of the key factors on cAMP/PKA signaling pathway,and Fluorescence Quantitative RT-PCR method was used to detect the mRNA expression of the key factors on NF-?B pathway.Results Compared with the normal group,the activity of MCs in the 20%,10%,5%,1.25%DHI groups were increased significantly(P<0.01),while the activity of MCs in the model control were decreased significantly(P<0.01).Compared with the model group,the activity of H/R MCs in 20%,10%,5%,2.5%DHI groups were increased significantly(P<0.01,P<0.05).Compared with the normal group,the apoptosis rate of MCs in the model control and 10%,5%DHI groups were increased significantly(P<0.01);Compared with model group,the apoptotic rate of MCs in verapamil and 20%,10%,5%DHI groups were decreased significantly(P<0.01,P<0.05).The results of ELISA,Western Blotting and Flurescent Quantitation RT-PCR showed that the model groups had a lower protein expression on AC,CREB,a lower mRNA expression on AC,CREB,PKA(P<0.01,P<0.05),and a higher protain expression on p38MAPK(P<0.01)when they were compared with normal group.Compared with the model group,the protein expression on AC,CREB,p38MAPK of each dose group of DHI increased in various degrees(P<0.01,P>0.05,P<0.05).20%DHI group had a higher mRNA expression on AC(P<0.05).20%DHI and 10%DHI groups had a higher mRNA expression on CREB,PKA(P<0.01,P<0.05).the mRNA expression of NF-?B,TNFR-1,TRADD,IkB?,IL-1? increased,and it had statistical difference compared with the normal group(P<0.01).each group of DHI had a lower mRNA expression on NF-?B,TNFR-1,TRADD,IkB?,IL-1?(P<0.01,P<0.05)compared with the model group,while those of RIPA had no difference(P>0.05).Conclusion DHI can increase the cell activity of MCs and protect H/R MCs.The protective mechanism of DHI on H/R MCs may be related with the pathway:(1)Protect the MCs by inhibiting apoptosis.(2)Protect the MCs through the promotion of energy metabolism by increasing AC,CREB,PKA protein expression and AC,CREB,p38MAPK mRNA expression in cAMP/PKA signaling pathway.(3)Protect the MCs by inhibiting inflammatory response through inhibiting the mRNA expressions of NF-?B,TNFR-1,TRADD,IkBa,IL-1? in NF-?B signaling pathway. |
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