Study On The Role And Mechanism Of The Macrophage Inflammation And Foam Cell Formation Mediated By Piperlonguminine And Its Derivatives

ObjectiveThe inflammatory foam cell model of atherosclerosis(AS)was established by stimulating THP-1 monocyte-derived macrophages with palmitate(PA).Subsequently,the model was used to assess the inhibitory effect of Piperlonguminine(GBN)and its two derivatives GBNT and GBNX on foam cell formation and inflammation.The effects of drugs on lipid metabolism regulatory genes and autophagy-NLRP3 inflammatory axis in foam cells were detected to explore the anti-AS mechanism of Piperlonguminine and its derivatives.MethodsThe effects of GBN,GBNT and GBNX on the viability of macrophages were detected by MTS,and the appropriate concentration was selected within the non-toxic range of the drug.Phorbolester(PMA)was used to induce THP-1 cells to differentiate into macrophages,and then PA was used to stimulate macrophages derived from THP-1 to establish an inflammatory foam cell model,which was divided into model group(Model group)and drug intervention group,and drug intervention groups were divided into positive control group(Sim group),GBN group(2?M),GBNT group(2?M)and GBNX group(2?M).After 24 hours of treatment,the supernatant and cells were collected and the changes of each index were detected.1.Oil red O staining was used to detect the accumulation of intracellular lipids in each group to explore the effect of GBN and its derivatives on the formation of foam cells.2.The content of intracellular cholesterol in each group was measured by enzymecolorimetry,and the ratio of cholesterol ester to total cholesterol was calculated to explore the effect of GBN and its derivatives on intracellular cholesterol esters.3.The expressions of scavenger receptors SR-A,LDLR,CD36 and cholesterol efflux transporters SR-BI,ABCA1 and ABCG1 mRNA in macrophages were detected by RT-PCR to explore the possible pathway of GBN and its derivatives in regulating ester metabolism.4.The contents of inflammatory cytokines IL-1 ?,TNF-? and IL-6 in macrophage supernatant and cells were detected by ELISA and RT-PCR to explore the effect of GBN and its derivatives on inflammation.5.The content of ROS in macrophages was detected by DCFH-DA fluorescence probe to explore the antioxidant effect of GBN and its derivatives.6.The protein expression levels of NLRP3,Caspase-1(P20),IL-1 ?,LC3 B,P62,SIRT3 and SOD2 were detected by WB to explore the possible mechanism of GBN and its derivatives in inhibiting foam cell inflammation.Results1.THP-1 cells were induced to differentiate into macrophages by stimulating THP-1cells with PMA of 200ng/mL for 72 hours.The adhesion rate of macrophages induced by this concentration was also high and the morphology was typical and intact.The results of oil red staining showed that stimulation of THP-1-derived macrophages with 20?mol/L PA for 24 hours could increase the number of intracellular lipid droplets and lipid content significantly,and these differences were statistically significant(P<0.01).The model of THP-1 macrophage-derived foam cells could be successfully established.2.MTS results showed that GBN,GBNT and GBNX had certain inhibitory effects on the proliferation of THP-1-derived macrophages,but the low concentration had little effect.GBN and its derivatives were non-toxic to THP-1-derived macrophages when the drug concentration of ?16?mol/L,so GBN,GBNT and GBNX with concentration ?16?mol/L were selected for follow-up experiments.3.The results of oil red staining showed that GBN,GBNT and GBNX could significantly reduce the accumulation of lipids in foam cells.The results of enzymeendpoint method showed that PA stimulation significantly increased the contents of total cholesterol(TC),free cholesterol(FC)and cholesterol ester(CE)in macrophages(P<0.001),and the ratio of CE/TC reached 54.6%.However,GBN,GBNT and GBNX could significantly decrease the intracellular CE/TC ratio(P<0.001,P<0.01),which were36.7%,38.4% and 39.2%,respectively.4.The results of real-time quantitative PCR showed that PA stimulation significantly increased the expression of scavenger receptors SR-A,LDLR and CD36 in THP-1 macrophages(P<0.001),while GBN,GBNT and GBNX significantly decreased the mRNA expression of SR-A,LDLR and CD36(P<0.001,P<0.01),and increased the mRNA expression of cholesterol efflux transporters SR-BI,ABCA1 and ABCG1 in macrophages(P<0.001,P<0.01,P<0.01).It can also be seen from the experimental results that GBN has the best effect on inhibiting lipid uptake by macrophages or promoting cholesterol efflux from macrophages.5.The results of ELISA and RT-PCR showed that PA stimulation significantly increased the expression of IL-1?,IL-6 and TNF-? in the culture supernatant and cells of THP-1 macrophages(P<0.001),while after GBN,GBNT and GBNX treatment,the expression of IL-1?,IL-6 and TNF-? in the culture supernatant and cells decreased significantly(P<0.001,P<0.01,P<0.05).Compared with the experimental results,it was found that the anti-inflammatory effect of GBN was better than that of its derivatives.6.The results of DCFH-DA probe method showed that the intracellular ROS level of THP-1 macrophages increased significantly after PA stimulation(P<0.001),and the intracellular ROS level induced by PA was significantly inhibited by GBN,GBNT and GBNX(P<0.001).7.The results of WesternBlotting showed that the expression of SOD2 in macrophages increased significantly after PA stimulation,and after treatment with GBN,GBNT and GBNX,the expression of SOD2 decreased significantly in GBN group and GBNT group(P<0.05),but there was no significant difference compared with Model group.Further study showed that compared with Control group,20 ?mol/L PA significantly increased the expression of SOD2 in macrophages,but when theconcentration of PA increased to 250 ?mol/L,the expression of SOD2 decreased significantly(P<0.05).8.WesternBlotting results showed that PA stimulation significantly increased the expression of NLRP3,Caspase1 and IL-1? in THP-1 macrophages(P<0.001,P<0.01).After treatment with GBN,GBNT and GBNX,the expression of NLRP3,Caspase1 and IL-1? in THP-1 macrophages was significantly decreased(P<0.001,P<0.01,P<0.05).9.WesternBlotting results showed that PA stimulation slightly increased the ratio of autophagy-associated protein LC3II/I in THP-1 macrophages,but there was no significant difference compared with Control group,at the same time,the expression of p62 was significantly increased(P<0.001).After treatment with GBN,GBNT and GBNX,the ratio of LC3II/I increased significantly and the expression of p62 decreased significantly in all groups(P<0.01).10.WesternBlotting results showed that PA stimulation slightly increased the expression of autophagy regulatory protein SIRT3 in THP-1 macrophages,but there was no significant difference compared with Control group,and the expression of SIRT3 in all groups increased significantly after treatment with GBN,GBNT and GBNX(P<0.05).11.The results of WesternBlotting showed that the expression of SIRT3 and the ratio of LC3II/I in macrophages treated with GBN,GBNT and GBNX were significantly higher than those in Control group(P<0.01).Conclusion1.THP-1 monocytes can differentiate into typical and intact macrophages after 72 hours of 200ng/mL PMA stimulation,and then differentiate into foam cells after 24 hours of 20?mol/L PA stimulation.2.GBN has no toxicity to THP-1 macrophages in the concentration range of0-16?mol/L,while GBNT and GBNX have no toxicity to THP-1 macrophages in the concentration range of 0-32?mol/L.3.GBN,GBNT and GBNX can inhibit the lipid uptake of macrophages by inhibiting the expression of scavenger receptors SR-A,LDLR and CD36,promote the expression of cholesterol efflux transporters SR-BI,ABCA1 and ABCG1,regulate the reversecholesterol transport process,reduce the accumulation of lipids in macrophages,and then inhibit the foam of macrophages.4.GBN,GBNT and GBNX can inhibit the expression and release of inflammatory cytokines IL-1 ?,IL-6 and TNF-? during PA-stimulated macrophage foaming,suggesting that GBN and its derivatives have anti-inflammatory effects,and its anti-inflammatory mechanism may be through the direct clearance of ROS,or by increasing the expression of SIRT3 to promote macrophage autophagy,and then inhibit the activation of NLRP3 inflammasome,so as to play the role of anti-AS.5.The inhibitory effect and anti-inflammatory effect of GBN on macrophage foaming are better than GBNT and GBNX.