| ObjectiveThe preliminary clinical results showed that Qingre Huoxue Decoction(QRHX)had the effect of inhibiting atherosclerosis(AS).Macrophages derived from monocytes are present in the whole stage of AS,and macrophage polarization plays a crucial role in plaque formation.In this study,combining experiments in vivo and in vitro,methods of high-performance liquid liquid-mass spectrometry,network pharmacological analysis,technology of molecular docking andRNA transcriptome sequencing,the underlying mechanism of QRHX on AS were explored from the perspective of macrophage polarization.It provides new targets and ideas for the intervention of ASCVD by way of QRHX.Methods1.Efficacy study(1)Sixty 8-week-old male ApoE-/-mice were randomly divided into blank group,model group,QRHX low-dose group,medium-dose group and high-dose group,and Lipitor group,with 10 mice in each group.The AS model of ApoE-/-mice was established by giving high fat diet(16 weeks),and the corresponding drug intervention was started at 8 weeks,and samples were collected at the end of 16 weeks.(2)Blood was collected from the abdominal aorta,and the levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C)were detected by Automatic Biochemistry Analyzer to evaluate the level of blood lipid.The levels of NLRP3,MCP-1 and TNF-?in serum of ApoE-/-mice were determined by Enzyme linked immunosorbent assay(ELISA).(3)The aorta was separated and lipid deposition was assessed by oil red O staining.Plaque size was assessed by HE staining;Injure components of ApoE-/-mice were assessed by Sirius red staining and immunohistochemical(IHC)staining(MOMA-2 and-SMA staining)in high-fat diet.The phenotypic expression of polarization M1(iNOS)/M2(Arg-1)in macrophages was detected by western blot(WB).2.Target prediction(1)The compound components in the QRHX were qualitatively detected by liquid chromatography-mass spectrometry(LC-MS).According to the pharmacological analysis of the combined network of selected components,the signal pathway in intervening AS of QRHX was predicted.The key proteins of the signaling pathway were preliminarily verified WB.(2)The components of QRHX were quantitatively analyzed to screen out the core active components,and the core components were molecularly docked with the genes in the signal pathway in(1)to conduct the secondary screening of the core genes.(3)In vitro experiment,Cell count kit(CCK 8)was used to screen the concentration range of interference cells in the solution of QRHX lyophilized powder.Mice derived macrophage cell line RAW264.7 was cultured.The expression of macrophage specific markers iNOS(M1 phenotype)and Arg-1(M2 phenotype)were qualitatively analyzed by immunofluorescence(IF)method.RT-qPCR was used to detect the mRNA expression levels of CCR7(M1 phenotype)and CD163(M2 phenotype)to determine the optimal concentration of the solution of QRHX to induce the polarization of RAW264.7 cells to M2phenotype.(4)ByRNA high-throughput sequencing,the core signal pathway of QRHX regulating macrophage polarization was obtained,and the genes in the pathway were verified by RT-qPCR.3.Verification of mechanism(1)Ppre-driven luciferase reporter plasmid was transfected to construct inhibitory plasmid.RAW264.7 cells were treated with heat-clearing and blood-activating formula combined with inhibition plasmid.(2)The mRNA levels of CCR7(M1 phenotypic marker)and CD163,FIZZ-1,YM-1,and Arg-1(M2 phenotypic marker)were detected by RT-qPCR.(3)The expression of iNOS(M1 phenotypic marker)and Arg-1(M2 phenotypic marker)were quantitatively analyzed by IF method.(4)The levels of IL-1?(M1-phenotype macrophage release),IL-4 and IL-10(M2-phenotype macrophage release)cytokines in cell supernatant were determined by ELISA.(5)WB was used to verify the target protein in the pathway.Results1.Efficacy study(1)ApoE-/-mice AS model was successfully constructed:the results of aortic oil red O staining indicated that the lipid deposition level of model group was increased compared with blank group(P<0.01);In HE staining,the plaques in the model group were more obvious than those in the blank group,and obvious fibrous caps were visible in the plaques.(2)The QRHX could improve the levels of blood lipids,inflammatory factors and stabilized plaques in ApoE-/-mouse AS model:The results of automatic biochemical analyzer showed that compared with the blank group,the levels of Tc,TG and LDL-c in model group were increased,but the levels of HDL-c were not significantly different.It is suggested that QRHX could decrease the levels of TC,TG and LDL-C.Compared with model group,NLRP3 level in QRHX medium-dose group decreased,and NLRP3 level in QRHX high-dose group and Lipitor group significantly decreased.The level of MCP-1 in QRHX medium-dose group was decreased,and the level of MCP-1 in QRHX high-dose group and Lipitor group was significantly decreased.The level of TNF-?in QRHX medium dose group,QRHX high dose group and Lipitor group was significantly decreased.QRHX could down-regulate the levels of inflammatory factors NLRP3,MCP-1 and TNF-?.The results of aortic oil red O staining indicated that the lipid deposition in the low-dose group of QRHX was decreased compared with the model group,and the lipid deposition in the medium-dose and high-dose groups of QRHX and Lipitor groups was significantly decreased compared with the model group.In HE staining,the plaques of QRHX high-dose group and Lipitor group were significantly reduced compared with model group.It is suggested that the formula for clearing heat and activating blood circulation has the effect of stabilizing plaque.(3)Macrophages were involved in the damage component of ApoE-/-mice induced by high fat diet,and the mechanism was related to the polarization of macrophages:Sirius red picric acid staining and?-SMA staining indicated that there was no significant difference between groups.MOMA-2 staining indicated that compared with the model group,the content of MOMA-2 in the groups of QRHX and Lipitor group was significantly decreased,suggesting that macrophages were involved in the intervention of AS by QRHX.WB results suggested QRHX of the medium and high dose and lipitor group compared with model group,expression of iNOS protein decreased significantly,QRHX medium dose group of Arg-1 protein expression increased.The expression of Arg-1 protein in the high dose of QRHX and Lipitor group were significantly increased,suggesting that the mechanism of QRHX's intervention on AS was related to the polarization of macrophages.2.Target prediction(1)Qualitatively detected compound components obtained by LC-MS method,combined with network pharmacological analysis,and verified by WB method,it was concluded that PI3K/Akt might be the key signal pathway of QRHX to interfere with AS.(2)The quantitative results in the QRHX indicated that baicalin and salvianolic acid B might be the core components of QRHX.Through molecular docking with PI3K/Akt pathway genes,it was found that baicalin and salvianolic acid B had the lowest binding free energy for docking with RXRA.(3)The concentration of QRHX lyophilized powder solution at 5-100?g/m L had no effect on the survival rate of RAW264.7 cells.The results of IF indicated that the positive rate of iNOS expression in LPS group was significantly higher than that in the blank group,and the positive rate of QRHX groups was lower than that in the LPS group.However,the expression of Arg-1 was low in the LPS group and the blank group,and the fluorescence expression was significantly increased when the concentration of QRHX lyophilized powder solution was 10?g/m L.RT-qPCR results showed that the expression of CCR7 in the LPS group was significantly higher than that in the blank group,and the expression of it in the QRHX group was decreased to different degrees,especially when the concentration of QRHX lyophilized powder solution was 10?g/m L.The expression of CD163 increased to different degrees in the groups of QRHX,and the most significant was when the concentration of QRHX lyophilized powder solution was 10?g/m L.In conclusion,QRHX lyophilized powder solution at the concentration of 10?g/m L has the most significant regulation on the polarization of macrophages.(4)RNA high-throughput sequencing was performed between the RAW264.7+LPS group and LPS RAW264.7+LPS+10?g/ml QRHX group,and differentially expressed threshold is set to:|log FC|>1 and P value<0.05.A total of 445 differentially expressed genes were collected,containing 258 down-regulated genes and 187 up-regulated genes.By KEGG analysis,33 signaling pathways such as NF-?B were obtained.Sixteen genes in the NF-?B pathway were verified by RT-qPCR,and the most significant differences were found between Cxcl3 and Ptsg2 genes.3.Verification of mechanism(1)Cxcl3-siRNA and Ptgs2-siRNA inhibitory plasmids were constructed by transfection with luciferase reporter plasmid,and the plasmids were successfully transfected.Compared with NC-siRNA,the mRNA expressions of Cxcl3-siRNA and Ptgs2-siRNA were significantly decreased(P<0.01),suggesting that the plasmid was successfully transfected.(2)RT-qPCR showed that compared with the LPS group,the levels of macrophage M1phenotypic target CCR7 were down-regulated in the LPS+Cxcl3-siRNA group,the LPS+Ptgs2-siRNA group,and the LPS+RXRA-siRNA group,and the levels of macrophage M2phenotypic target Fizz-1,YM-1,and Arg-1 were up-regulated to varying extent,suggesting that macrophages were polarized to M2 phenotype after inhibition of Cxcl3,Ptgs2,and Rx RA genes.Compared with the LPS+Cxcl3-siRNA+10?g/m L QRHX group,the LPS+Ptgs2-siRNA+10?g/m L QRHX group and the LPS+RXRA-siRNA+10ug/m L QRHX group compared with the LPS+Ptgs2-siRNA group and the LPS+RXRA-siRNA+10?g/m L QRHX group compared with the LPS+RXRA-siRNA group,the levels of macrophage M1phenotypes target CCR7 were down-regulated,and the levels of macrophage M2phenotypes target CD163,Fizz-1,YM-1 and Arg-1 were up-regulated to different degree.It is suggested that the addition of heat-clearing and blood-activating formula can promote the polarization of macrophages to M2 phenotype.(3)The results of the IF suggested that iNOS and Arg-1 were expressed in the nucleus and cytoplasm of RAW264.7.Compared with LPS+Cxcl3-siRNA+10?g/m L QRHX group,LPS+Ptgs2-siRNA+10?g/m L QRHX group and LPS+RXRA-siRNA+10?g/m L QRHX group,the positive rate of iNOS of M1 phenotype of macrophages was decreased,suggesting that the addition of the QRHX inhibited macrophages from polarizing to M1phenotype.Compared with LPS+Cxcl3-siRNA+10?g/m L QRHX group,LPS+Ptgs2-siRNA+10?g/m L QRHX group and LPS+RXRA-siRNA+10?g/m L QRHX group,the positive rate of Arg-1 was increased,suggesting that the addition of QRHX promoted the polarization of macrophages to M2 phenotype.(4)The results of ELISA showed that Compared with LPS+Cxcl3-siRNA+10?g/m L q RHX group,LPS+Ptgs2-siRNA+10?g/m L QRHX group,LPS+RXRA-siRNA+10?g/m L QRHX group compared with LPS+Ptgs2-siRNA group,and LPS+RXRA-siRNA+10?g/m L QRHX group compared with LPS+RXRA-siRNA group,the levels of inflammatory cytokines IL-1?released by the M1 phenotype of macrophages were down-regulated,while the levels of cytokines IL-4 and IL-10 released by the M2 phenotype of macrophages were up-regulated.It is suggested that the addition of QRHX inhibits the polarization of macrophages to M1 phenotype and promotes the polarization of macrophages to M2phenotype.(5)WB showed that LPS+Cxcl3-siRNA+10?g/m L QRHX group was compared with LPS+Cxcl3-siRNA group,LPS+Ptgs2-siRNA+10?g/m L QRHX group was compared with LPS+Ptgs2-siRNA group,LPS+RXRA-siRNA+10?g/m L QRHX group was compared with LPS+RXRA-siRNA group,phosphorylated proteins p-PI3K and p-Akt in PI3K/Akt pathway,phosphorylated proteins p-p50 and p-p65 in NF-?B pathway were compared with LPS+RXRA-siRNA group.In addition,the relative expression level of phosphorylated protein p-IKK was down-regulated in PI3K/Akt and NF-?B pathways,suggesting that the addition of QRHX can more effectively down-regulate the expression of key proteins in PI3K/Akt and NF-?B pathways,and play an inhibitory role in PI3K/Akt and NF-?B pathways.Conclusion1.The results of animal experiments showed that after the intervention of QRHX in ApoE-/-mouse AS model,it played the role of regulating lipid and stabilizing plaque and reducing the expression of inflammatory factors.Macrophages are involved in the damage of high fat diet to ApoE-/-mice,and the mechanism may be related to the polarization of macrophages.2.Sixty-eight active components of QRHX were found by qualitative analysis of LC-MS,and combined with network pharmacological analysis method,it was predicted that the anti-AS effect of QRHX might be realized through PI3K/Akt pathway,and was verified at the protein level.Combined with quantitative analysis of QRHX and molecular docking technology,the results suggested that RXRA gene might be the core gene of QRHX in the prevention and treatment of AS.3.The results ofRNA transcriptome high-flux sequencing suggested that QRHX might exert anti-inflammatory effects by mediating NF-?B to regulate macrophage polarization,and Cxcl3 and Ptgs2 are two key genes in this pathway.4.Cell experiment results showed that QRHX could promote the polarization of macrophages to M2 phenotype and reduce the expression of inflammatory factors,and the specific mechanism may be realized by mediating RXRA,Cxcl3 and Ptgs2 genes to regulate PI3K/Akt and NF-?B pathways.5.QRHX might inhibit inflammation and play an anti-AS role by inhibiting PI3K/Akt and NF-?B signaling pathways to regulate the polarization of macrophages to M2 phenotype. |
PDF Full Text Request