Effects Of Monomers Active Metabolite Of Panax Notoginseng Saponins On Foam Cell Formation And Its Mechanisms

Notoginseng, the roots of Panax notoginseng F.H.Chen, has been used as health product and herbal remedies in cardiovascular diseases for more than 1,000 years in traditional Chinese medicine. Present studies on Saponins of panax notoginseng, the major effective ingredient extracted from Panax Notoginseng, showed that it exhibited various anti-atherogenic activities, including its ability to reduce the proliferation of vascular smooth muscle cell, anti-hyperlipemia, protection against artery injury, et al.Nevertheless, there are still some questions which caused the restriction of PNS studies could not be answered, because of the complicated components of PNS. So, the studies focus on the effects of monomers respectively became hot recently. But the previous studies in vivo on the monomers of PNS showed that some substances which was considered as the main active component formerly could not exist in serum, such as Rb1. After oral administration of red Ginseng extracts, the plasma concentration of ginsenosides in healthy volunteers was examined by EIA-HPLC, and the result showed that Rb1 was not detected in the blood. Akao reported that when Rb1 (200 mg·kg-1) was administered orally to germ-free rats, not any metabolite was detected in the plasma, and most of the Rb1 administered was recovered from the intestinal tract, indicating poor absorption of Rb1. These studies indicated that after oral administration of mice with Rb1, neither intact Rb1 nor its intermediate derivatives was detected in blood , but only the final metabolite could be detected in blood. The results above suggested that Rb1 might play its activities via its final metabolite after oral administration. So in this study, we tried to find the effects on AS in vitro of the metabolites of Rb1 and Rg1, two main components of PNS (Rb1 29.7%, Rg1 28.5%), which named NG701 and NG801 by our team.Atherosclerosis (AS) is a fundamental pathological change which results in some serious cardiovascular diseases, such as stroke, coronary artery disease, hypertension etc. The certain pathogenesy of AS is still undiscovered, while it is generally accepted that macrophage foam cells are the characteristic feature of atherosclerotic plaques. During foam cell formation, modified lipoproteins enter cells by receptor mediated uptake and excessive neutral lipids are stored as lipid droplets, creating a typical foamy appearance. The formation of foam cells are considered as the initiation of AS, which also can influence the progression and destination of the pathological change.In current study, the cholesterol content of macrophage foam cells and the expression of CD36, ABCA1, HMG-CoA Reductase mRNA expression were examined in order to investigate the probably mechanism of the anti-atherogenic effects of the active metabolites of PNS, NG701 and NG801.Methods:1. NR8383cells were incubated with ox-LDL for 24h to establish foam cell model. Different concentrations of NG701,Rb1,NG801,Rg1 were given for intervention. The cellular lipid accumulation was examined by oil red staining.2. The cellular contents of total cholesterol (TC) were detected by enzymic methods.3. The levels of ABCA1,LXRα,HMG-CoA,CD36 mRNA were determined by real time-PCR.Results:1. Under light microscope, cells of the control group were round or fusiform and there was no oil red-staining; Because of uptaking amount of lipid, oil red-staining positive cells were filled with macrophages and the shapes were round or irregular after co-cultured with ox-LDL. When NG701 group administration concentration was 10,30μmol/L, cells color turned light and the shape became normal.2. When administration was 3,10μmol/L, NG801 cells showed no significant difference in color with model group by oil red"O"staining. While at 30,100μmol/L, cells color turned light and the shape became normal.3. The TC of model group in foam cell were significantly higher than control group (P<0.01). Compared with model group, total cholesterol content in cells showed dose dependent decreasing when NG701 administration. The concentration of 10μmol/L NG701 reduced the TC level in foam cells.4. At the administration concentration of 30μmol/L NG801, the TC decreased significantly compared with model group (P<0.05); At the concentration of 100μmol/L NG801, TC decreased even greater (P<0.01) compared with model group. 5. Compared with model group, when the administration concentration of NG701 group was 3μmol/L, the cells expression of CD36, LXRα, ABCA1 and HMG-CoA mRNA were not altered. While the concentration reached 10μmol/L, it down-regulated the expression of CD36 mRNA (P<0.01) and HMG-CoA mRNA (P<0.01), and up-regulated the expression of LXRαmRNA (P<0.05) and ABCA1 mRNA (P<0.01); When the concentration reached 30μmol/L, it down-regulated the expression of CD36 mRNA (P<0.01) and HMG-CoA mRNA (P<0.01), and up-regulated the expression of LXRαmRNA (P <0.01) and ABCA1 mRNA (P<0.01).6. Compared with model group, when the concentration of NG801 reached 3,10μmol/L, the mRNA expression of CD36, LXRα, ABCA1 and HMG-CoA were not altered. When concentration reached to 30μmol/L, it down-regulated the CD36 mRNA(P<0.01), up-regulated the expression of LXRαmRNA(P<0.05) and ABCA1 mRNA(P<0.01), and had no impact on HMG-CoA mRNA; When the concentration reached to 100μmol/L, it down-regulated CD36 mRNA(P<0.01)and HMG-CoA mRNA(P<0.01), up-regulated LXRαmRNA(P<0.01)and ABCA1 mRNA(P<0.01).Conclusions:1. NG701,NG801 produced a dose-dependent reduction of TC in the macrophage- derived foam cells. After treated by the NG701,NG801, the red staining materials in the foam cells were decreased, and the cell morphology was gradually to be nomal. Moreover, the activity of NG701,NG801 was higher than the prototype drugs, that was Rb1,Rg1.2. NG701 and NG801 could up-regulate the expressions of ABCA1 mRNA and LXRαmRNA to degrade the cholesterol in the foam cells through down-regulating the expressions of CD36 mRNA and HMG-CoA mRNA. This result could decrease the accumulating of cholesterol in the macrophage, and promote outflowing of cholesterol. Above all, NG701 and NG801 had effects on the foam cells formation from every links.