Explore The Anti-atherosclerotic Mechanism Of Xiongshao Capsule From The Perspective Of Reverse Cholesterol Transport

Objective:To explore anti-atherosclerosis mechanism of Xiongshao Capsule(XC)on reversing cholesterol transport pathway.Methods:(1)The target genes of Chuanxiong and Chishao in XC were predicted and analyzed on BATMAN-TCM platform.Then,the similarity between n Compounds of XC-Target"and"Known Drug-Target"was calculated,and the potential drug-target interaction was scored.Targets with score(>20)were used as potential target genes,based on functional items and organisms.A network of Compound-Target-Biological Pathway-Disease was constructed for investigating the biological functions and pathways of potential targets of XC.(2)RAW264.7macrophages were treated with80mg/L oxidized low-density lipoprotein for24h in order to obtain foam cells.The foam cells were intervened with drug-containing serum derived from rats.The macrophages were assigned to control group(normal macrophages),model group(80mg/L ox-LDL),normal serum(NS)group[10%normal rat serum(NRS)],normal serum control group(80mg/L ox-LDL and10%NRS),simvastatin drug-containing serum(SDS)group(80mg/L ox-LDL and10%SDS),low-dose group of Xiongshao Compound drug-containing serum(XCDS)(80mg/L ox-LDL and10%XCDS),high-dose group of XCDS(80mg/L ox-LDL and20%XCDS).The distribution of cholesterol in cells were evaluated by Oil red O stain.The contents of total cholesterol(TC)and free cholesterol(FC)were assessed using commercial kits.(3)The effects of different concentrations of tetramethylpyrazine and paeoni florin on the proliferation of foam cells were detected by CCK-8,respectively.According to the effect of different compatibility between tetramethylpyrazine and paeoniflorin on intracellular cholesterol,the best combination dose was determined.Subsequently,RAW264.7cells were divided into5groups:control group,model group,tetramethylpyrazine group,paeoniflorin group,and jointed group inoculated on96-well plates,and intervened with different drugs.The distribution of intracellular lipids in foam cells treated with the effective components of Xiongshao Capsule(ECXC)was observed by oil red O staining.RAW264.7cells iwere divided into6groups:control group,model group,apoAl group,tetramethylpyrazine group,paeoniflorin group and j ointed group.They were intervened by different drugs respectively,and the cholesterol efflux capacity of ECXC on foam cells were observed by fluorescence labelling cholesterol.RAW264.7cells were divided into5groups:control group,model group,tetramethylpyrazine group,paeoniflorin group,and jointed group inoculated in60mm petri dish,intervened with different drugs,and cells were collected.The effect of ECXC on the expression of RCT related proteins such as peroxisome proliferator-activated receptor gamma(PPARy),liver X receptor alpha(LXRa),adenosine triphosphate binding cassette transporter Al(ABCA1)adenosine triphosphate binding cassette transporter G1(ABCG1),scavenger receptor B1(SR-B1)in foam cells were observed by Western blot and PCR,respectively.The contents of tumor necrotic factor alpha(TNF-a),interleukin 1beta(IL-β)and monocyte chemokine-1(MCP-1)in cell culture medium of different groups were detected by Elisa kit.(4)DIA analysis was used to detect the dynamic protein changes of foam cells derived from RAW264.7treated with ECXC.Results:(1)A total of965target genes were obtained from BATMAN-TCM platform related to 110compounds in XC.Several components of XC were involved in regulating the expression of PPAR gamma,ABCA1,ABCG1,apoAl and other related RCT proteins,and affecting the metabolism of CCL2,IL-1β,TNF-a and CD36.The pharmacological activity of XC involves 13gene targets related to AS,including3-hydroxy-3-methylglutaric coenzyme A reductase(HMGCR),human heme oxygenase-1(HMOX1),arachidonic acid-15-lipoxygenase(ALOX15),microsomal triglyceride transfer protein(MTTP),peroxisome proliferator-activated receptor gamma(PPARG),liver X receptor alpha(NR1H3),peroxisome,proliferation factor activated receptor delta(PPARD),monocyte chemokine1(CCL2),arachidonate15-lipoxygenase B(ALOX15B),carboxylesterase 1(CES1),nuclear receptor subfamily 1H group2(NR1H2),thrombokine Ⅱ(F2)and estrogen receptor1(ESR1),involving cholesterol transmermbrane tra nsport,coagulation,cholesterol ester metabolism,inflammation,lipid and oxidative modification,cell adhesion and other processes.(2)The cell morphology and staining results of the cells in model group induced by 80 ug/ml ox-LDL met the foam cell standard.A large number of lipid droplets were found in all groups except for control group,and there was no significant difference in lipid staining between the groups.Compared with NS,there were not obvious difference in TC of SDS and XCDS(P>0.05),while more FC existed in SDS(P<0.05),yet less in XCDS(P<0.05).Compared with the control group,the contents of TC and FC in model group,NS group,NSC group,SDS group and high-dose group of XCDS increased(P<0.05),while the contents of TC and FC in low-dose group of XCDS did not increase significantly(P>0.05),while the contents of FC increased(P<0.05).Compared with the model group,the TC levels in the low-dose group of XCDS decreased(P<0.05),but there was no significant difference in the FC levels,and the TC and FC levels increased between the NS group,the NSC group,the SDS group and the high-dose group of XCDS(P>0.05).Compared with the NSC group,the TC content in the low-dose group of XCDS decreased(P<0.05),and the FC content had no significant difference(P>0.05),but the TC and FC concentration in the serum group of simvastatin and the high-dose group of XCDS had no significant difference(P>0.05).(3)Compared with the control group and model group,the effect of tetramethylrazine and paeoniflorin on the proliferation of foam cells was not statistically significant(P>0.05).Compared with the control group,the levels of TC and FC in the model group increased significantly(P<0.05).Compared with model group,TC content of 40mg/L tetramethylpyrazine group and 80mg/L tetramethylpyrazine group decreased significantly(P<0.05);TC content of different concentration of tetramethylpyrazine(10,20,40mg/L)group and different concentration of paeoniflorin(5,10，20,40,80mg/L)group decreased significantly(P<0.05);the FC content of 40mg/L tetramethylpyrazine group and 40mg/L tetramethylpyrazine group with different concentrations of paeoniflorin(5,10,20,40,80 mg/L)decreased(P<0.05),while the FC content of other groups did not decrease significantly(P>0.05).Oil red Ostaining showed that the cells in the control group were not stained.Compared with the control group,the cells in the model group were round like,and the cell morphology was vacuolar,and a large number of red lipid droplets were found in the cells,which met foam cell standards.Compared with model group,intracellular lipid deposition in tetramethylpyrazine group,paeoniflorin group and jointed group decreased significantly,and tetramethylpyrazine group,and paeoniflorin group showed a small amount of red lipid droplets,but no obvious lipid staining was found in the jointed group.Compared with the control group,the concentration of TNF-α,IL-β and MCP-1in the model group increased significantly.Compared with model group,the concentration of IL-1β and MCP-1 in Tetramethylpyrazine group decreased significantly(P<0.05),the concentration of IL-1p in paeoniflorin group decreased(P<0.05),and the concentration of TNF-a,IL-1β and MCP-1 in jointed group decreased significantly(P<0.05).There was no fluorescent cholesterol deposition in the control group.Compared with the control group,a large amount of fluorescent cholesterol was accumulated in the model group.The intracellular fluorescent cholesterol in apoA1 group,tetramethylpyrazine group,paeoniflorin group and jointed group decreased in different degrees,and the jointed group decreased significantly.Compared with the model group,the cholesterol efflux rate of apoAl group,tetramethylpyrazine group,paeoniflorin group and jointed group increased significantly(P<0.05).Compared with apoAl group,the cholesterol efflux rate of tetramethylpyrazine group and jointed group increased significantly(P<0.05).Compared with the control group,the expression of SR-B1protein increased in the model group(P<0.05),PPAR gamma protein decreased significantly in tetramethylpyrazine group(P<0.05),SR-B1 protein increased in paeoniflorin group(P<0.05),and the expression of ABCA1,ABCG1,SR-B1,PPAR gamma and LXRalpha protein increased significantly in the jointed group(P<0.05).Compared with the model group,the expression of SR-B1protein in tetramethylpyrazine group decreased significantly(P<0.05),while the expression of ABCAl,ABCG1,PPAR gamma and LXRalpha protein in jointed group increased significantly(P<0.05).Compared with the control group,the expression of PPARgamma and LXRalpha in the model group decreased significantly(P<0.05),and the expression of ABCA1,ABCG1,PPARgamma and LXRalpha in the jointed group increased significantly(P<0.05),while the expression of SR-B1 had no statistical difference(P>0.05).Compared with the model group,the expressions of ABCA1 and SR-B1 in tetramethylpyrazine group were significantly increased(P<0.05).The expressions of ABCA1,ABCG1,SR-B1,PPAR gamma and LXRalpha in the jointed group were significantly increased(P<0.05).(4)Nine samples were analyzed by DIA technology,and the results showed that there were 324 and 142 differentially expressed proteins in model group/control group and jointed/model group,respectively.KEGG pathways in model group/control group were mainly concentrated in DNA replication,pentose and glucuronide,AMPK infiltration pathway,fatty acid metabolism,glutathione metabolism,carbon metabolism and central carbon metabolism in cancer.The related pathways of jointed group/model group focused on apoptosis,butyric acid metabolism,peroxisome,pentose phosphate,purine metabolism and insulin resistance.Further analysis showed that 24common differential proteins were screened out in the two comparison groups.Poly[ADP-ribose]polymerase 2(PARP2)may be a new target of lipid metabolism related to the effective components of XC.The expression of PARP2 increased in model group,while decreased in jointed group treated with ECXC.Conclusion:(1)XC involves 13 gene targets related to AS,including cholesterol transmembrane transport,coagulation reaction,cholesterol ester metabolism,inflammation reaction,lipid oxidation modification,cell adhesion and so on.The predicted results are consistent with the previous studies of XC.At the same time,it provides some new potential target genes and research ideas for XC in the future research field of anti-atherosclerosis.(2)The drug-containing serum derived from rats itself contains a large number of lipids which can cause cholesterol accumulation,even froth in RAW 264.7 cells,and belies the effect of XC on the cholesterol efflux in foam cells,it may not be suitable used in the study of cholesterol outflow in foam cells from RAW 264.7 macrophages.(3)XC can activate the PPAR gamma/LXR alpha pathway,upregulate the expression of ABC A1,ABCG1 and SR-B1,promote cholesterol efflux in foam cells,inhibit the release of TNF-alpha,IL-1 beta and MCP-1 and prevent macrophage froth.(4)A new target of XC in lipid regulation,PARP2,was found by proteomic analysis.The ECXC may promote intracellular lipid efflux by inhibiting the expression of PARP2 and up-regulating the expression of ABCA1.