American Ginseng Saponin On Coronary Heart Disease Insulin Sensitivity And Its Mechanism

IR is the common physiopathologic mechanism of metabolic disorders such as coronary heart disease, diabetes, hypertension and dyslipidemia. Many studies have proven that IR could contribute to the occurance and development of coronary heart disease by secondum hyperinsulinemia, metabolic disorder of glucose and lipid, hypertension, functional disorder of vascular endothelial cell, hyperplasy of smooth muscle cell, platelet activation, disorder of blood clotting and fibrinolysis system, and so on. So IR is one of CHD risk factors. Improvement of IR could contribute to relieve the glucose-lipid metabolism dysfunction and vascular lesion. To prevent and cure CHD, we not only need to relieve vesscle lesion and enhance plaque stability, but also need to improve IR.Panax quinquefolius saponin (PQS) is extracted from leaf and stem of American ginseng. The contents of saponin, amino acids and mineral elements in stem and leaf are higher greatly than in the root. A multicentre and double-blind study organized by Ministry of Public Health has proven that PQS not only can regulate lipid metabolism and improve the blood supply of ischemic myocardium, but also can decrease plasma glucose in 1999. Then how can PQS accomplish the actions simultaneously? Is there any common mechanism? Is the effect of PQS on regulating glucose-lipid metabolism relative to its improvement of insulin resistance?In our study, the insulin resistant model of 3T3-L1 adipocytes is established in vitro by elevated palmitic-BSA level. Focusing on glucose and lipid metabolism and insulin-signaling transduction, we observe the effect of PQS on 2-deoxy-[3H]-D-glucose transport, the translocation of the GLUT-4 from intracellular sites to the cell surface, tyrosine phosphorylation of insulin receptor and IRS-1, serin phosphorylation of PKB, lipid degradation and glucose consumption, the mRNA levels of CAP and PPAR-y, which will provide theoretical evidence for the treatment of metabolic disorders such as coronary heart disease and diabetes. The clinical study is to investigate the effect of PQS on blood glucose and lipids, insulin sensitivity and explore the relationships between the change of insulin sensivity and the pattern of syndrome of patients with CHD and pathoglycemia.PART I : Basic Studies[ I A] Objective To observe the effect of PQS on the expression of PPAR-y(PPAR-y) in the differentiation of 3T3-L1 preadipocyte. Methods 3T3-L1preadipocytes were cultured. Confluent predaipocytes were treated with ifiMdexamethasone, lQMg/ml insulin and 0.5mM IMX for 48h, followed by standardmedium IMDM culture with lOjMg/ml insulin. Oil red O staining was used to evaluatethe conversion process of 3T3-L1 adipocytes on day 0, 5, 7 and day 9. The peroxisomeproliferator activated receptor-y mRNA was detected by RT-PCR. Results Theundifferentiated group had no expression of PPAR-y mRNA. However, RT-PCRshowed great expression of PPAR-y mRNA in the differentiated group, PQS groups andmetformin group. Comparing with the differentiated group, there was no significantchange in PQS groups and metformin group (P>0.05 ) , only slight increase trend. Inthe differentiated group, PQS group and metformine group, the level of PPAR-y mRNAon day9 was lower than those on day 5 and day 7. Conclusion There is highexpression of PPAR-y mRNA in the differentiation process of 3T3-L1 preadipocytes.PQS and metformine can slightly increase PPAR-y gene expression in thedifferentiation process, which may be correlated with insulin sensitivity improvement.[IB] Objective To observe the effects of PQS on glucose transportation,GLUT-4 translocation and CAP mRNA expression in insulin resistance model ofadipocytes. Methods The insulin resistant model of differentiated 3T3-L1adipocytes was established in vitro by elevated palmitic-BSA level. The uptake of2-deoxy-[3H]-D-glucose was used to observe glucose transportation. The GLUT-4translocation was observed by immunofluorescence. The CAP mRNA was detected byRT-PCR. Results The uptake of 2-deoxy-[3H]-D-glucose in model group(2.143±0.472) was significantly lower than in control group (4.993+0.917) (P<0.01) .The amount of uptake in meformin group was 4.180±0.717, 3.942±0.492 in high-dosePQS group, 3.160±0.450 in middle-dose PQS group, which were greatly higher thanthose in model group ( P<0.01 ) ( P<0.01 ) ( P<0.05 ) . The level of2-deoxy-[3H]-D-glucose uptake showed no significant differences between low-dosePQS group and model group (P>0.05) . In the absence of insulin, most GLUT-4 incontrol group was present in the cell interior. However, following the insulinstimulation for 30 minutes, the GLUT-4 localized in the cell interior decreased, whileincreased relatively around the cell membrane. In model group, there were no significant differences between insulin absence and insulin presence. Treatment with metformin or PQS, insulin stimulation resulted in the same influence as in control group. RT-PCR showed that the level of CAP mRNA in model group was significantly lower than in control group (P<0.05) . Treatment with metformin, high-dose and middle-dose PQS could greatly increase the expression of CAP mRNA (P<0.05) . There was no significant change in low-dose PQS group and model group (P>0.05) . Conclusion PQS can accelerate the expression of CAP mRNA, GLUT-4 translocation to plasma membrane and glucose transportation, which may be correlated with PQS improving insulin resistance of adipocytes. Is the function that PQS can accelerate GLUT-4 translocation and glucose transportation a pharmacal direct effect or a secondery effect of regulating glucose-lipid metabolism to decrease FFA? Which need to be confirmed by further study.[ I C] Objective To observe the effects of PQS on insulin signal transduction and glucose-lipid metabolism in the insulin resistant model of 3T3-L1 adipocytes. Methods The insulin resistant model of differentiated 3T3-L1 adipocytes was established in vitro by elevated palmitic-BSA level. After induction of insulin resistance, cells were treated with metformin or PQS for 2 days. Then the phospholation of signal proteins was detected by western-blot. The amount of glucose which disappeared from the culture medium after incubation with drugs for 24h was determined as glucose consumption of the cells. Colorimetry was used to observe the effects of PQS on the lipolysis induced by TNF-a. Results The tyrosine phosphorylation of insulin receptor and IRS-1 and Ser473 phosphorylation of PKB were lower in model group than in control group. Compared with model group, low-dose PQS group showed no obvious difference. But treatment with high-dose, middle-dose PQS or Metformin could increase the level of phospholation of insulin receptor, IRS-1 and PKB to different extent. The amount of glucose consumption in model group (5.250±2.671) was significantly lower than in control group(14.133±1.305) (P<0.01) . The amount of glucose consumption in meformin group was 11.807±1.358, 10.784±2.373 in high-dose PQS group, 10.217±1.237 in middle-dose PQS group, 9.984±2.006 in low-dose PQS group, which were greatly higher than those in model group (P<0.01) . Upon TNF-a treatment, the concentration of FFA in culture medium is 2.479±0.597, predominantly higher than that in controlgroup (1.320±0.538 ) (P<0.05) . The concentration of FFA in metformin group was 1.210±0.566, 1.105±0.631 in high-dose PQS group, 1.108±0.260 in middle-dose PQS group, 1.201 ±0.593 in low-dose PQS group, which were greatly lower than those in TNF-a group (P<0.05, P<0.05, P<0.01, P<0.05) .But there were no significant changes in all groups treated with metformin and PQS. Simultaneously, the action of PQS appeared a dose-dependent tendency. Conclusion When FFA-induced insulin resistance occurs in adipocytes, the level of tyrosine phosphorylation of insulin receptor and IRS-1 and Ser473 phosphorylation of PKB would decrease, which inhibits the insulin signal transduction and finally depresses the uptake and consumption of glucose. Treatment with high-dose, middle-dose PQS or Metformin could increase the level of phospholation of insulin receptor, IRS-1 and PKB, which may be contributed to utilize glucose. Is the action of PQS promoting insulin signal transduction a pharmacal direct effect or a secondery effect of regulating glucose-lipid metabolism to decrease FFA? Which need to be confirmed by further study. On the other hand, PQS not only can accelerate the glucose consumption, but also can depress lipolysis of adipocytes induced by TNF-a, which is beneficial to regulate the glucose-lipid metabolism and improve insulin resistance in adipocytes.PART II: Clinical StudyObjective To investigate the effect of PQS on blood glucose and lipids, insulin sensitivity and explore the relationships between the change of insulin sensivity and the pattern of syndrome of patients with CHD and pathoglycemia. Methods 84 patients with CHD and pathoglycemia were randomly divided into PQS group (43 cases) and control group(41 cases) treated with routine western medicine. On the basis of standard medication of mordern medcine, patients in PQS group were treated with PQS for 4 weeks. Detect the levels of FPG, FINS, TC, TG, HDL and LDL both before and after treatment. ISI=1/ (FPGFINS). Homa-IR =(FPGxFINS)/22.5,Homa-8=20xFINS/(FPG-3.5). Results The availability rates of angina, electrocardiogram and pattern of syndrome between the two groups showed no significant difference. After treatment, FPG decreased predominantly in both groups (P<0.01). Furtheremore, FPG in PQS group decreased more than in control group. In both groups, ISI slight increased slightly, and Homa-IR decreasded trivaly, but the deviation was not significant (P>0.05). Between PQS group and control group, ISI andHoma-IR showed no significant deviation. Homa-8 increased markedly after treatmentin PQS group, and the level of Homa-6 was higher obviously in PQS group than incontrol group. In PQS group, TC and LDL decreased greatly after treatment and werepredominantly lower than in control group (P<0.05) (1.17±0.54 and 1.42±0.49respectively). But TG and HDL showed no significant difference after treatment.Treatment with routine western medicine reduced no obvious change in TC, TG, LDLand HDL. The results of pattern of syndrome study showed that: ?In the excessivesyndromes on patients with CHD and pathoglycemia, ISI decreased gradiently in theorder of qi-stagnancy and blood stasis, cold accumulation and blood stasis, phlegmatichygrosis and blood stasis. There were great difference between the syndrome ofphlegmatic hygrosis and blood stasis and the syndrome of qi-stagnancy and blood stasis.Homa-IR increased gradually in the order of qi-stagnancy and blood stasis, coldaccumulation and blood stasis, phlegmatic hygrosis and blood stasis. Homa-IR washigher obviously in the syndrome of phlegmatic hygrosis and blood stasis than in thesyndrome of qi-stagnancy and blood stasis (P<0.05). (2)In the deficiency syndromes, ISIdescended gradiently in the order of the qi deficiency syndrome, the yin deficiencysyndrome, the qi and yin deficiency syndrome, and yang deficiency syndrome, but therewas no predominant difference among 3 groups. Homa-IR and Homa-8 in the threegroups showed no significant deviation (P>0.05). Conclusion Comparing with routinewestern medicine treatment, combining PQS seemed to show superiority in loweringFPG on the patients with CHD and pathoglycemia. It also could decrease the levlels ofTC and LDL significantly, which is superior to routine western medicine treatment.Combining PQS on routine western medicine treatment maybe improve 8-cell function,which need to be confirmed by a large sample study. Analysis to pattern of syndromeindicated that: In excessive syndromes, IR of phlegmatic hygrosis and blood stasissyndrome was most obvious and the insulin sensivity was poorest. At the same time, ISIdecreased gradiently in the order of qi-stagnancy and blood stasis, cold accumulationand blood stasis, phlegmatic hygrosis and blood stasis. In the deficiency syndromes, ISIof the qi and yin deficiency syndrome and the yang deficiency syndrome was lowest,which could indicate that IR became more and more serious with progression of disease.But relationships between the change of insulin sensivity and the pattern of syndrome ofpatients with CHD and pathoglycemia need to be verified by lage sample data.SUMMARYIn conclusion, we assumed that the effect of PQS on regulating glucose-lipid metabolism is relative to its improvement of insulin resistance. So we established the insulin resistant model of 3T3-L1 adipocytes in vitro by elevated palmitic-BSA level. Focusing on insulin-signaling transduction, we observed the effect of PQS on tyrosine phosphorylation of insulin receptor and IRS-1, Ser473 phosphorylation of PKB, 2-deoxy-[3H]-D-glucose transport, the translocation of the GLUT-4 from intracellular sites to the cell surface, lipid degradation and glucose consumption, the mRNA levels of CAP and PPAR-y. Finally, we not only found that PQS could increase the tyrosine phoshpolation of insulin-receptor and IRS-1 and the Ser473 phoshpolation of PKB, encourage CAP genetic transcription, promote GLUT-4 translocation, increase glucose transportation, accelerate glucose uptake, improve insulin resistance, increase insulin sensitivity of adipocytes, but also found that PQS could inhibit lipolysis induced by TNF-a and have a tendency to increase PPAR-y gene transcription slightly. The above results indicate that PQS could increase insulin sensitivity and regulate glucose-lipid metoblism by promoting insulin signal transduction. Is the post-receptor action of PQS a pharmacal direct effect or a secondery effect of regulating glucose-lipid metabolism to decrease FFA? Which need to be confirmed by further study. The clinical study showed that: Comparing with routine western medicine treatment, combining PQS seemed to show a superior tendency in lowering FPG on the patients with CHD and pathoglycemia. It also could decrease the levlels of TC and LDL significantly, which is superior to routine western medicine treatment. Combining PQS on routine western medicine treatment maybe improve 6-cell function, which need to be confirmed by a large sample study. Analysis to pattern of syndrome indicated that: In excessive syndromes, IR of phlegmatic hygrosis and blood stasis syndrome was most obvious and the insulin sensivity was poorest. At the same time, insulin sensitivety decreased gradiently in the order of qi-stagnancy and blood stasis, cold accumulation and blood stasis, phlegmatic hygrosis and blood stasis. In the deficiency syndromes, ISI of the qi and yin deficiency syndrome and the yang deficiency syndrome was lowest, which could indicate that IR became more and more serious with development of disease. But relationships between the change of insulin sensivity and the pattern of syndrome of patients with CHD and pathoglycemia need to be verified by lage sample data.