Effects Of Carvacrol On Macrophage Foam And Autophagy In RASMCs

BackgroundAtherosclerosis is a progressive disease characterized by the accumulation of lipids and large arterial fibrous elements.Chronic inflammation plays an important role in the development of atherosclerosis.Activated leukocytes,endothelial cells and macrophages produce proinflammatory cytokines including IL-1?,IL-6,and TNF-a as well as anti-inflammatory cytokines such as IL-10.From the Chinese herbal medicine,plants to find effective and toxic side effects of small anti-atherosclerosis drug targets,can become a new century,domestic and foreign scholars to develop new directions for anti-lipid drugs.It has been shown that carvacrol has a wide range of pharmacological properties and minimal toxicity to human normal cells.A number of experimental studies have shown that carvacrol has anti-inflammatory,anti-atherosclerotic intimal hyperplasia effect.But the specific mechanism of action is not clear.This study was to investigate the effect of caramel on the autophagy induced by ox-LDL and to explore its possible molecular mechanism.Method1,in vitro culture of oxidized low density lipoprotein-induced TPH-1 cells into foam cells,and then treated with different concentrations of carvacrol(terminal concentration of 0.1,1.0,5.0,10 uM)treatment of macrophages RAW246.7 cells 24 hours,and the anti-proliferative effect and induced apoptosis were observed with the increase of the concentration of carvacrol.THP-1 cells were treated with PRMI-1640 medium and 10%non-mycoplasma neonatal calf serum as supplemental medium in 37'C,5%C02 cell incubator.The cells were cultured in logarithmic growth phase.THP-1 cells were cultured with 100 nM phorbol myristone(PMA)for 24 h to induce differentiation into macrophages.After the medium was replaced with Ox-LDL with different concentration gradient,,Making it into foam cells.The apoptosis of TPH-1 cells induced by oxidized low density lipoprotein was observed by Hoechst33258 staining.Apoptosis of RAW246.7 cells was detected by Hoechst33258 staining.The P38-MAPK signal pathway and ERK/MAPK signal pathway specific blocking agent U0216 were used to block the ERK/MAPK signal pathway,and the cell activity and expression were detected by blocking the P38-MAPK signaling pathway specific SB203580.The experiment is grouped as follows:(1)carvacrol-treated group were treated with different components of carvacrol for 27 and 48 h,the control group and the drug group were used to observe the positive rate and stability of carvacrol to RAW264.7 cells The effect of carvacrol on MAPK signaling pathway of phosphorylated protein and its total protein expression was detected by Western Blot in RAW264.7 cells.(2),the inhibitor group:RAW264.7 cells were treated with 20 MU0126A(ERK1/2 specific inhibitor)and 10 M SB203580B(P38 specific inhibitor)for 48 h,24 h,the blank control was not The effect of RAW264.7 on the inflammation and plaque stability of RAW264.7 cells was observed.(3),carvacrol and inhibitor combination group:To observe the specific inhibition of MAPK signal transduction pathway on the effect of carvacrol on RAW264.7 cell inflammation.The combination of 5 uM carvacrol,10 M U0126 and 10 M SB203580 was applied to RAW264.7 cells for 24 h,and the inhibitor was added to CAR for 1 h.4,through the oil red 0 staining shows the state of intracellular lipid droplets,detection of caramel to inhibit the formation of foam cells.Cells were inoculated into culture plates,after oxldl induced modeling,and the detection of different carvacrol concentration inhibited the level of foam cells.5,To observe whether carvacrol inhibits ox-LDL-induced LOX-1 expressionThe total RNA of the cells was subjected to RT-PCR to detect LOX-1 protein levels in different concentrations of Carvacrol.6,Western Blot analysis of carvacrol on oxidized low-density lipoprotein-induced TPH-1 cells into foam cells in the foam and NFkB protein in a variety of phosphorylation formsWestern Blot method includes 1,filling and loading 2,electrophoresis 3,transfer film 4,closed 5,immune responseTo observe the effect of carvacrol on Ox-LDL-induced inflammatory factors.The levels of TNF-a?IL-1 beta and IL-6 in the supernatant of the culture supernatant were detected by ELISA after 48 h of THP-1 macrophages treated with corrosine 1 uM,5 uM and 10 uM of Carvacrol.,.To observe the effect of carvacrol on autophagy induced by oxidized low density lipoprotein.The oxidized low density lipoprotein-induced TPH-1 cell foam and autophagy of RAVSMCs were detected by electron microscopy and Western blot.(1)Select the best 150-180g SD rats,tissue culture vessels were divided into blank control group(1%double antibody serum-free DMEM basal),carvacrol group(MCP1 final concentration of 100ng/ml).While retaining some of the fresh blood vessels in the liquid nitrogen cryopreservation.Western blot was used to detect the expression of autophagy-related proteins mTOR,p62,LC3? and LAMP-2.Results1,MTT detection of different concentrations of carvacrol(terminal concentration of 0.1,1.0,5.0,10 uM)treatment of macrophages RAW246.7 cells 24h after the increase in the concentration of carvacrol its proliferation activity did not change significantly,Compared with the control group,there was no significant difference between the two groups(P>0.05).After 24 hours of oxldl stimulation with carvacrol(0.1,1.0,5.0,10mM)(P>0.05).There was no significant difference in the proliferative activity(P>0.05)2,Carvacrol in vitro experiments on the macrophage cell line RAW246.7 cell foam has a significant inhibitory effect Hoechst33258 nuclear staining analysis showed that different concentrations of carvacrol treated RAW246.7 cells after 24 hours.with the increase of concentration,number of cells reduce gradually,show enrichment dense solid form or particles state on the number of apoptotic cells increased,the obvious cell debris.,The results of nuclear staining showed that the apoptosis of RAW246.7 cells was not changed after blocking the ERK/MAPK signaling pathway,but the apoptosis of RAW246.7 cells was significantly decreased after blocking the p38/MAPK signaling pathway.3,carvacrol inhibit the droplets of foam cells:by microscopic-observation,control group cytoplasm in the red fat insignificant and small.There were a lot of red lipid droplets in the cytoplasm of the ox-LDL treated cells,and the red fat droplets accounted for more than 50%of the cell volume.Compared with the control group,the intracellular lipid droplets were decreased in the intracellular lipid droplets of the ox-LDL group,and the number of intracellular lipid droplets decreased with the increase of the concentration of carvacrol(CAR).4,The expression of LOX-1 in carvacrol group was significantly lower than that in control group(P<0.05).And the higher the concentration of carvacrol,the lower the Lox-1 protein level.The level of LOX-1 protein in Carvacrol group was significantly different from that in ox-LDL group(p<0.05).However,there was no significant difference between Carvacrol luM group and ox-LDL group(p>0.05).5,The levels of TNF-a,IL-6 and IL-1 beta in the supernatant of the cells were significantly higher than those in the control group(P<0.05).The expression of TNF-a,IL-1? in the supernatant was significantly higher than that in the control group(P<0.05 vs control).The levels of TNF-a,IL6 and IL-1 beta decreased with the increase of concentration of Carvacrol,and there was significant difference compared with the ox-LDL group(p<0.05 vs ox-LDLgroup)6,The expression of TNF-a,IL-1 beta and IL-6 in LPS-induced inflammatory cytokines induced by carbohydrate was significantly higher than that of other groups after treatment with LPS group.(P<0.01).The levels of TNF-a,IL-1 beta and IL-6 in LPS group were significantly higher than those in other groups,and the levels of TNF-a Increased(p<0.01).7,The expression of mTOR and p62 in monocyte-macrophage induced by ox-LDL was significantly higher than the control group(P<0.05),and the expression of mTOR protein in ox-LDL group was significantly higher than that in control group(P<0.05)Level.Compared with ox-LDL group,the expression of mTOR protein was decreased by three doses of carvacrol,which was significantly lower than that of ox-ldl group in 10uM CAR group.The expression of p62 protein in each group was consistent with mTOR.Compared with ox-LDL group,the expression of p62 protein in ox-LDL + mid-dose group and ox-ldl + carvacrol high-dose group decreased,and the expression of p62 protein was statistically significant(P<0.05).There was no significant differences in The expression of LC3 II protein in ox-LDL group and control group.Compared with ox-LDL group,carvacrol increased the expression of LC-3? protein,and the expression of LC3II in ox-LDL+ middle-dose group was up-regulated,and the high-dose of ox-ldl + carvacrol was up-regulated and the value was statistically significant(P<0.05).Conclusion(1).Carvacrol had obvious inhibitory effect on the foaming of macrophage line RAW246.7 cells in vitro.The apoptotic effect of carvacrol was related to p38-MAPK signal pathway.;(2).Carvacrol inhibited the lipid droplet content of foam cells;(3).Carvacrol inhibited ox-LDL-induced LOX-1 expression;(4).Carvacrol inhibited the expression of inflammatory factors in foam cells induced by ox-LDL;(5).Carvacrol inhibits the activation of NF-?B by ox-LDL;(6).Carvacrol inhibits the expression of inflammatory cytokines induced by LPS in foam cells;(7)Carvacrol can decrease the expression of mTOR,p62 and LAMP-2 in aortic smooth muscle cells,and the effect of carvacrol on the expression of mTOR and p62 of monocyte-macrophage induced by ox-LDL.In summary,carvacrol(CAR)may play a role in the prevention of atherosclerosis by regulating the proliferation of macrophages,inhibiting the formation of foam cells,and regulating autophagy-lysosomal metabolism of LC3.