Effect Of Rhodiola On Expression Of Adrenomedullin In Ischemia Rat Hearts And HUVECs Exposed To Hypoxia

Part I Effect of Rhodiola on expression of AM and CRLR in ischemia rat hearts during angiogenesis period after acute myocardial infarctionObjective:1. To compare the angiogenic effect of Rhodiola and Salidroside in ischemia rat hearts using a rat acute myocardial infarction model. 2. To investigate the effect of Rhodiola and Salidroside on expression of AM and CRLR in ischemia rat hearts. 3. To investigate the effect of Rhodiola on levels of plasma AM in rats after acute myocardial infarction.Methods:1. Rhodiola powder was prepared according to a detailed protocol.From every 1kg crude Rhodiola crenulata,211.59g powder was made.And the Salidroside content of Rhodiola powder was 0.83%,analyzed by High Performance Liquid Chromatography-ultraviolet Detection.6%(w/w) Rhodiola solution was the optimal concentration.All Salidroside solution was prepared with the same content of Salidroside compared with Rhodiola solution.2. Five groups of male Wistar rats were studied:Fifteen rats ligated as AMI model were fed with natural salt (N group), Rhodiola solution(R group) or Salidroside solution(S group) from 7 days before to 7 days after operation.Another 6 rats were randomized to sham group (SII group) and normoxia control group(C group).All rats were phlebotomized and sacrificed 7days after operation.3. Immunohisto chemistry assay(IHC) for detection of CD31,realtimt quantitative PCR with SYBR Green for detection of mRNA levels of AM,CRLR, Western Blot analysis for detection of their protein levels and ELISA for detection of plasma AM levels.Results:1. Results of IHC expressed as MVD showed that R group had the highest expression of CD31,S group took second place and NS group had lowest expression at the border of infarction place.2. Results of PCR were expressed as RQ (2-(ΔΔCT).R group had the highest mRNA expression of AM and CRLR. S group took second place with AM and CRLR.But the difference between R group and S group on expression of AM was not statistically significant. The difference between R group and S group on expression of CRLR was not statistically significant either. SH and C groups had lowest expression of AM and CRLR.3. Results of Western were normalized toβ-actin. R group had the highest protein expression of AM and CRLR. S group took second place with AM and CRLR. But the difference between R group and S group on expression of CRLR protein was not statistically significant. SH and C groups had lowest expression of AM and CRLR.4. Compared with SH group, R group,S group and N group had higher plasma AM levels.And the plasma AM levels of R group,S group and N group were similar.Conculsions:Pretreatment of Rhodiola or Salidroside can promote angiogenesis in rat ischemia heart and elevate the mRNA and protein expression of AM and CRLR.And the effect of Rhodiola was better. Compared with N group, Rhodiola can not raise the level of plasma AM in rats after AMI. Part II Effect of Rhodiola on expression of AM and CRLR in human umbilical vein endothelial cells (HUVECs) exposed to hypoxiaObjective:1. To compare the angiogenic effect of Rhodiola and Salidroside in HUVEC. 2. To investigate the effect of Rhodiola and Salidroside on expression of AM and CRLR in HUVEC exposed to hypoxia. 3. To investigate whether Rhodiola promates expression of AM and CRLR via a HIF-1α-dependent pathway.Methods:Step one:Choose the best dose of Rhodiola to have proper growth effect on HUVEC by CCK8 array. Step two:Four groups of HUVECs were studied, Normoxia control group (N group), Hypoxia control group (H group), Hypoxia and Rhodiola treatment group (H+R group), Hypoxia and Salidroside treatment group (H+S group). Realtimt quantitative PCR with SYBR Green for detection of mRNA levels of AM,CRLR, and Western Blot analysis for detection of their protein levels. Step three:1) Three groups of HUVECs were studied, Normoxia control group (N group), Hypoxia control group (H group), Hypoxia and Rhodiola treatment group (H+R group). Realtimt quantitative PCR with SYBR Green for detection of mRNA levels of HIF-la, and Western Blot analysis for detection of their protein levels.2) Optimizing siRNA transfection conditions by using the FAM+negative control siRNA (FAM-siRNA) and the positive control siRNA (GAPDH siRNA). Three groups of HUVECs were studied; N group, H group, and H+R group.Each group had four sub-groups, blank control group, transfection control group, negative control siRNA group and HIF-1αsiRNA group. Realtimt quantitative PCR with SYBR Green for detection of mRNA levels of HIF-1α,AM and CRLR, and Western Blot analysis for detection of their protein levels.Results:Step one:Rhodiola and Salidroside both had cheering effect on cellular proliferation, and the former is better.10ug/ml Rhodiola is the proper dose for HUVEC to grow, and the optimal treatment length was 24h. Step two:H+R group had the highest expression of AM and CRLR, either in mRNA or in protein level. H+S group took second place, and H group also had higher mRNA and protein expression of AM and CRLR than N group.The differences were all statistically significant. Step three:1) H+R group had the highest expression of HIF-la, either in mRNA or in protein level. H group also had higher mRNA and protein expression of HIF-la than N group.2)The final siRNA concentration of 60nM was optimal.No difference was found between blank control, transfection control group and negative control siRNA group for HIF-1α,AM or CRLR mRNA/protein expression.After transfection of HIF-lasiRNA, mRNA and protein expression of HIF-1α,AM and CRLR were remarkablely attenuated.After transfection of HIF-la siRNA,the expression of HIF-1α,AM and CRLR mRNA/protein in H+R group was mildly higher than in H group,N group had lowest expression.But the differences between these groups were not statistically significant.Conculsions:Rhodiola and Salidroside had cheering angiogenic effect.They could promote the expressions of AM and CRLR in HUVECs exposed to hypoxia. Rhodiola had better effect than Salidroside. Rhodiola promotes expressions of AM and CRLR via a HIF-1α-dependent pathway in HUVECs exposed to hypoxia.