Ginsenoside Rb1 Regulates Macrophage Polarization And Stable Atherosclerotic Plaque And Its Mechanism

BackgroundAtherosclerosis(AS)is a chronic inflammatory disease of the arterial wall.Inflammation contributes to robust increases in atherosclerotic plaque size and complexity.Smooth muscle cells,macrophages and endothelial cell are involved in the inflammation of plaques,among which macrophages play a vital role.As a highly plastic and versatile cell,macrophages can differentiate into two antagonistic subtypes: classically activated by interferon-gamma(IFN-?)or lipopolysaccharide(LPS),known as M1;or alternatively activated by interleukin IL-13 or IL-4,known as M2.The two phenotypes of macrophage play a different role in inflammation through production of pro-inflammatory or anti-inflammatory cytokines,depending on microenvironmental stimuli.The M1 macrophages are potent defensive cells that kill microorganisms and produce pro-inflammatory cytokines,such as tumor necrosis factor-?(TNF-?),IL-6 and IL-1?.On the contrary,M2 macrophages produce anti-inflammatory factors such as IL-10,transforming growth factor-?(TGF-?),which alleviate inflammatory responses and contribute to angiogenesis and tissue repair.Recent studies have demonstrated that M2 macrophages may play a protective role in atherosclerosis.Early atherosclerotic lesions are infiltrated by more M2 while M1 prevail in advanced atherosclerotic lesions in Apo E KO mice.Recent studies reveal that the macrophage phenotype is a better indicator of plaque outcome.Ginsenoside Rb1 is a representative component of panaxadiol saponins which is an herbal medicine that is used widely in East Asian countries.Previous studies have demonstrated its powerful pharmacological effects,including anti-cancer,anti-inflammation,lipid-lowering properties,anti-obesity activities,the ability against ischemia/reperfusion injury and endothelial cell apoptosis.Rb1 has been shown powerful potential in anti-inflammation.Rb1 administration reduced levels of pro-inflammatory cytokines in obese mice fed a high-fat diet.Given the role of macrophage polarization in inflammatory response,so we conclude that Rb1 may skew the M1/M2 balance towards a more preferable phenotype that reduces inflammatory response and increases plaque stability.Objective:1.To observe the effects of Rb1 on atherosclerotic plaque stabilization.2.To explore the underlying the mechanisms how Rb1 affects atherosclerotic plaque stabilization.Methods: 1.Isolation of peritoneal macrophages C57BL/6 mice(8-10 weeks old)were injected intraperitoneally with 1 m L 3% Thioglycollate Broth(fluka)to recruit macrophages to peritoneal cavity.Three days post-injection,peritoneal macrophages were collected by mouse peritoneal cavity using cold PBS.The cell pellets were obtained after centrifugation at 800 rpm for 5 min.The cells were incubated in 1640 RPMI supplemented with 10% FBS and 1% antibiotics for 3 h and washed three times to remove non-adherent cells.2.Cell culture RAW264.7 cells were obtained from the American Type Culture Collection(ATCC,VA,USA).Cells were cultured in culture medium(Dulbecco's modified Eagle's medium,10% fetal calf-serum)at 37°C humidified incubator under 5% CO2.RAW264.7 were divided into six groups: control,LPS,LPS+Rb1(10u M,20 u M,40 u M,80 u M).3.Western Blot method: Detecting the expression of i NOS,Arg1,p-stat6 and stat6.4.Flow Cytometry RAW264.7 cells were washed,blocked and then incubated for 30 min with APC-conjugated anti-mouse CD206 using the appropriate isotype controls.The stained cells were acquired on a BD Accuri flow cytometer(BD Biosciences)and analyzed with Flow Jo software(Tree Star,Inc.).5.Cytokine detection Levels of MMP-9,IL-10,IL-4 and IL-13 in culture supernatants were measured by enzyme-linked immunosorbent assays(ELISA)following the manufacturer's protocol.6.The aortic pathological examination: To observe the fat content of plaque by oil red,to observe the Collagen content of plaque by Sirius Red–stained,to observe the MOMA-2 and ?-SMA content of plaque by immunohistochemical method,evaluate the stability of the plaques.Stability coefficient:(collagen area + smooth muscle cell area)/(oil red area + macrophages area).Results1.Rb1 dose dependently inhibited the expression of the i NOS(M1 marker),meanwhile Arg-1(M2 marker)level was increased by Rb1 at the dose of 20 and 40 u M,compared with the LPS-stimulated macrophages.In addition,the percentage of M2 phenotype(CD206+)cells of Rb1 group was much higher than that of the LPS-stimulated group by flow cytometric analysis.Rb1 suppressed the inflammation in macrophages induced by LPS as demonstrated by a significant decrease in pro-inflammatory cytokine levels of MMP-9,while promoted the production of anti-inflammatory cytokines of IL-10.2.The amount of IL-4 and IL-13 in supernatant was significantly increased after treated with Rb1 for 24 h.The effects of Rb1 on macrophage polarization into the M2 phenotype were partly attenuated by IL-4 and IL-13 neutralizing antibodies.3.The level of p-STAT6 expression after treatment of 20 ?M Rb1 was approximately 2.0-fold higher than that in the LPS-stimulated group.Treatment with STAT6 inhibitors(leflunomide)could markedly suppress the M2 polarization of macrophages induced by Rb1.4.7 weeks after the administration of Rb1,the content of lipids and macrophages was significantly decreased in atherosclerotic plaques,but that of collagen and SMCs was significantly increased.Consequently,the vulnerability index of plaque in Rb1-treated mice was significantly reduced compared to the control group.MMP-9 positive staining was decreased significantly in the group of Rb1.5.The number of MOMA-2+ i NOS+ double positive macrophages in atherosclerotic lesions was significantly decreased in the Rb1 group compared with the control group,especially in the macrophage-rich lesion.Meanwhile,the number of double positive MOMA-2+Arg-1+ macrophages was significantly increased in the atherosclerotic lesions of Rb1 group.Conclusions: 1.Rb1 induces macrophages M2 polarization through secretion of IL-4 and/or IL-13.2.STAT6 plays an important role in macrophages M2 polarization induced by Rb1.3.Rb1 enhances atherosclerotic plaque stabilization through promoting differentiation of macrophages into an M2 phenotype.