| Objective:To discuss Chai Hu Shu Gan San combined with BMSCs transplantation’s protective effect on the myocardium of the AMI rats, and to discuss the effect mechanism about PI3K/Akt signaling pathway for further study.Method:1. Theoretical study:According to the discourse of past dynasties doctor about the etiology, pathogenesis, treatment of "thoracic obstruction","cardialgia" and "angina pectoris", and the latest progress about the bone marrow mesenchymal stem cells as the treatment of AMI patients, to explore the theory foundation of Chai Hu Shu Gan San combined with BMSCs transplantation’s protective effect on the myocardium of the AMI rats.2. Experimental study:Part1:The whole bone marrow adherent method and the density gradient centrifugation method were adopted for BMSCs separation and culture. SD rats were randomly divided into6groups:group A:sham group; group B:AMI group; group C: BMSCs group; group D:CHSG-L group (low concentration decoction of Chai Hu Shu Gan San by intragastric administration and BMSCs transplantation); group E:CHSG-M group(middle concentration decoction of Chai Hu Shu Gan San by intragastric administration and BMSCs transplantation); group F:CHSG-H group (high concentration decoction of Chai Hu Shu Gan San by intragastric administration and BMSCs transplantation). All rats were administrated by intragastric administration for7days after BMSCs transplantation, and were euthanized at30minute later after the last time. The rats’ serum was used to detect myocardial enzyme, SOD and MDA by the biochemical method, while detect HIF-la by ELISA method. The myocardial tissue at the edge of the myocardial infarction area was used to observe the myocardial morphology by HE staining, and to detect VEGF by immunohistochemical method.Part2:The whole bone marrow adherent method and the density gradient centrifugation method were adopted for BMSCs separation and culture. SD rats were randomly divided into6groups:group A:sham group; group B:AMI group; group C: BMSCs group; group D:MBC group (Chai Hu Shu Gan San by intragastric administration and BMSCs transplantation); group E:MBL group (LY294002(PI3K inhibition) by intraperitoneal injection and BMSCs transplantation); group F:MBCL group (LY294002by intraperitoneal injection, BMSCs transplantation and Chai Hu Shu Gan San by intragastric administration). All rats were administrated by intragastric administration for7days after BMSCs transplantation, and were euthanized at30minute later after the last time. The myocardial tissue at the edge of the myocardial infarction area was used to detect myocardial apoptosis by TUNEL method; to detect Bcl-2, Bax and caspase-9by immunohistochemical method; to detect total Akt, total GSK-3β,p-Akt and p-GSK-3β by western bolt method; to detect Akt mRNA and GSK-3βmRNA by RT-PCR method.Results:1. Theoretical study:Bad mood could influence the development of the disease early. Chai Hu Shu Gan San as an effective prescription of relieving the depressed liver, could be used as a basic prescription of treatment of "cure cardio by hepar". BMSCs transplantation was an immense potential clinical impact for curing AMI. Chinese herb extracts and Chinese herbal compound prescription played a role in mobilization, migration, proliferation and differentiation of stem cells, which could improve the therapeutic efficacy in BMSCs transplantation for acute AMI.2. Experimental study:Part1:1. Under optical microscope, the cells are spindle-shaped, polygon shaped, consistent in the shape, manifesting with the "swirl-like" growth. The cells have good homogeneity, high purity and meet the experimental requirements.2. The results of HE staining indicated that:To compare with the model group, group C to F could reduce the degree of myocardial cell necrosis, reduce the myocardial interstitial hemorrhage and alleviate soakage of inflammation cell; while increase the number of capillaries after AMI.3. To compare with the model group, group C to F could decrease myocardial enzyme CK, CK-MB in rats’ serum, the difference was statistically significant (P<0.01); increase the content of HIF-la, the difference was statistically significant (P<0.05); increase the protein expression of VEGF in myocardial cells, the difference was statistically significant (P<0.05). The effect of group F was more prominent. So, the protective effect of high concentration of Chai Hu Shu Gan San (1.52g crude drugs per mL) combined with BMSCs transplantation was better.Part2:To compare with the model group, group C and group D could increase the expression of Akt mRNA, raise the level of Akt phosphorylation, the difference was statistically significant (P<0.05); could decrease the expression of the downstream of GSK-3(3mRNA and raise the level of GSK-3J3phosphorylation to reduce the activity of GSK-3P, the difference was statistically significant (P<0.05); could increase the expression of Bcl-2while decrease the expression of Bax, Caspase-9, the difference was statistically significant (P<0.05); It also could decrease the myocardial apoptosis index to inhibit apoptosis in cardiac myocytes, the difference was statistically significant (P<0.05). The effect of group D was more prominent. After using LY294002, the expression of Akt mRNA and the level of Akt phosphorylation of group E and F was decreased, the difference was statistically significant compared with group D (P<0.05). To compare with group D, the expression of GSK-3(3mRNA was increased and the level of GSK-3β phosphorylation was decreased, the activity of GSK-3β was raised, the difference was statistically significant (P<0.05). The expression of Bcl-2was decreased while the expression of Bax and Caspase-9was raised; the myocardial apoptosis index was raised, so the effect of inhibiting apoptosis in cardiac myocytes was weakened, the difference was statistically significant compared with group D (P<0.05).Conclusion:1. Chai Hu Shu Gan San combined with BMSCs transplantation could improve myocardial tissue pathology morphology in rats, reduce leakage of the myocardial enzyme CK and CK-MB, increase the activity of SOD while inhibit MDA synthesis to reduce the metaboilic level oxygen radical, raise the count of HIF-1α and promote the VEGF synthesis of myocardial cells. The protective effect was better when Chai Hu Shu Gan San was at concentration of1.52g crude drugs per mL.2. Chai Hu Shu Gan San (1.52g crude drugs per mL) combined with BMSCs transplantation could inhibit apoptosis in cardiac myocytes of AMI rats. The mechanism might be related to activate the PI3K/Akt/Bcl-2signaling pathway and PI3K/Akt/GSK-3β signaling pathway. |
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