| Purpose: Taking Apo E-/-AS mice and RAW264.7 macrophages as the research object and macrophage glycometabolism reprogramming as the main starting point,this paper discusses the molecular mechanism of Huayu Qutan(Phlegm-dissipating & Statis-removing)formula in intervening the inflammatory reaction to exert influence on AS by regulating the macrophage glycometabolism reprogramming characterized by tricarboxylic acid cycle(TCA)reset and pentose phosphate pathway(PPP)activation.Material and method:Experiment 1: 24 Apo E knockout(Apo E-/-)mice were randomly divided into model group,Huayu Qutan formula group and simvastatin group fed with high-fat diet for 12 weeks.Eight C57BL/6J mice were fed with common feed as the blank group while mice in the other two groups were given gavage with corresponding drugs.The blank group and model group were given the same amount of normal saline.After 4 weeks,the contents of serum triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)were detected by automatic biochemical analyzer;Hematoxylin eosin(HE)staining was used to observe the morphological changes of aortic plaque;The level of inflammatory factors in the mice’s serum(IL-6,IL-1β ? TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA);The colocalization of aortic macrophages with proline hydroxylase(PHD),and glucose-6-phosphate dehydrogenase(G6PD)was observed by immunofluorescence;Protein and m RNA expression in aortic inflammatory factors(IL-1β ? IL-6 ? TNF-α)was detected by Western blot and real-time fluorescence quantification Q-PCR;Protein and m RNA expression in the factors related to macrophage glycometabolism reprogramming pentose phosphate pathway(G6PD?p38MAPK?NF-κB)in aortic macrophages of Apo E-/-AS mice was detected by Western blot and realtime fluorescence quantification Q-PCR;Protein and m RNA expression in the factors related to macrophage glycometabolism reprogramming tricarboxylic acid cycle reset(SHD?PHD?HIF-1α)in aortic macrophages was detected by automatic protein blot quantitative analysis system(WES)and real-time fluorescence quantification Q-PCR.Experiment 2: RAW264.7 macrophages were divided into control group,model group(induced by LPS),blank drug-contained serum group,Huayu Qutan formula drug-contained serum group,succinate dehydrogenase(SDH)inhibitor group(dimethyl malonate),Huayu Qutan formula drug-contained serum intervention succinate dehydrogenase inhibitor group.Enzyme linked immunosorbent assay(ELISA)was used to detect the contents of inflammatory factors(IL-6?IL-1β?TNF-α)in the first four groups;succinic acid(SA)kit was used to detect the content of SA in control group,model group,Huayu Qutan formula drugcontained serum group,dimethyl malonate group and Huayu Qutan formula drug-contained serum intervention dimethyl malonate group;Western blot and real-time fluorescence quantification Q-PCR were used to detect the protein and m RNA expression in the factors related to macrophage glycometabolism reprogramming tricarboxylic acid cycle reset(SHD?PHD?HIF-1α)of macrophages in the control group,model group,Huayu Qutan formula drugcontained serum group,dimethyl malonate group and Huayu Qutan formula drug-contained serum intervention dimethyl malonate group;enzyme linked immunosorbent assay(ELISA),Western blot and real-time fluorescence quantification Q-PCR were used to detect the content,protein and m RNA expression level of the inflammatory factors(IL-1β? IL-6?TNF-α)in the control group,model group,Huayu Qutan formula drug-containing serum group,dimethyl malonate group and Huayu Qutan formula drug-containing serum intervention dimethyl malonate group.Experiment 3: RAW264.7 macrophages were divided into control group,model group(induced by LPS),Huayu Qutan formula drug-contained serum group,glucose 6-phosphate dehydrogenase inhibitor group(RRX-001),Huayu Qutan formula drug-contained serum intervention glucose 6-phosphate dehydrogenase inhibitor group.Western blot and real-time fluorescence quantification Q-PCR were used to detect the protein and m RNA expression macrophage in the glycometabolism reprogramming pentose phosphate pathway activation related factors(G6PD ? p38 MAPK ? NF-κB)of macrophages in control group,model group,Huayu Qutan formula drug-containing serum group,RRX-001 group and Huayu Qutan formula intervention RRX-001 group;enzyme linked immunosorbent assay(ELISA),Western blot and real-time fluorescence quantification Q-PCR were used to detect the content,protein and m RNA level of inflammatory factor(IL-1)in the control group,model group,Huayu Qutan formula drug-contained serum group,RRX-001 group and Huayu Qutan formula drug-contained serum group intervention RRX-001 group.Results:Experiment 1:1.Blood fat level: compared with the blank group,the contents of serum TC,TG and LDL-C in the model group were significantly higher;Compared with the model group,the contents of serum TC,TG and LDL-C in Huayu Qutan formula group and simvastatin group decreased significantly;The differences were statistically significant(P<0.05 or P<0.01).2.Pathology of aortic plaque: compared with the blank group,the intima of aorta of mice in the model group was thickened,with narrowing lumen,and a large number of atherosclerotic plaques formed;Compared with the model group,the aortic plaque of mice in Huayu Qutan formula group and simvastatin group was significantly smaller.3.Serum inflammatory level: compared with the blank group,the level of serum IL-6?IL-1β?TNF-α in the model group increased significantly;Compared with the model group,the level of serum IL-6 ?IL-1β ? TNF-α in mice of the Huayu Qutan formula group and simvastatin group decreased significantly;The differences were statistically significant(P<0.05 or P<0.01).4.Colocalization of CD68 and PHD,CD68 and G6PD: compared with the blank group,the colocalization of CD68 and PHD in the model group decreased,while the colocalization in Huayu Qutan formula group and simvastatin group increased significantly;Compared with the blank group,the colocalization of CD68 and G6 PD increased significantly in the model group,and decreased in varying degrees in the Huayu Qutan formula group and simvastatin group.5.Protein and m RNA expression of aortic inflammatory factors: compared with the blank group,the expression level of IL-1β?IL-6?TNF-α in the model group increased significantly(P<0.01);Compared with the model group,the expression level of IL-1β ?IL-6 ?TNF-α in Huayu Qutan formula group and simvastatin group decreased significantly;The differences were statistically significant(P< 0.05 or P<0.01).6.Protein and m RNA expression of macrophage glycometabolism reprogramming TCA reset related factors: compared with the blank group,the relative expression of SDH and PHD in the model group decreased significantly,and the expression level of HIF-1 α increased significantly;Compared with the model group,the expressions of SDH and PHD in Huayu Qutan formula group and simvastatin group were significantly higher,and the expression of HIF-1 α decreased significantly;The differences were statistically significant(P<0.01).7.Protein and m RNA expression of macrophage glycometabolism reprogramming PPP activation related factors: compared with the blank group,the expression of G6 PD,p38MAPK and NF-κB in the model group increased significantly;Compared with the model group,the expression of G6 PD,p38MAPK and NF-κB in Huayu Qutan formula group and simvastatin group decreased significantly;The differences were statistically significant(P<0.01).Experiment 2:1.Macrophage inflammation level: compared with the control group,the level of IL-1β? IL-6? TNF-α in the model group increased significantly;Compared with the model group,the level of IL-1β? IL-6?TNF-α contained in Huayu Qutan formula drug-contained serum group decreased significantly;The differences were statistically significant(P<0.01).2.Succinic acid content: compared with the control group,the SA content in the model group increased significantly;Compared with the model group,the content of SA in Huayu Qutan formula drug-contained serum group decreased significantly;Compared with the model group,the content of SA in dimethyl malonate group increased significantly;Compared with dimethyl malonate group,the content of SA in Huayu Qutan formula drug-contained serum intervention dimethyl malonate group decreased significantly;The differences were statistically significant(P<0.01).3.Protein and m RNA expression of macrophage glycometabolism reprogramming TCA reset related factors: compared with the control group,the relative expression of SDH and PHD in the model group decreased significantly,and the relative expression of HIF-1α was significantly increased;Compared with the model group,the relative expressions of SDH and PHD in Huayu Qutan formula drug-containing serum group were significantly higher,and the relative expression of HIF-1α decreased significantly;Compared with the model group,the relative expressions of SDH and PHD in dimethyl malonate group decreased significantly,the relative expression of HIF-1α was significantly increased;Compared with the dimethyl malonate group,the relative expressions of SDH and PHD in Huayu Qutan formula drugcontained serum intervention dimethyl malonate group were significantly higher,and the relative expression of HIF-1α decreased significantly;The differences were statistically significant(P<0.01).4.Protein and m RNA expression of macrophage glycometabolism reprogramming TCA reset inflammatory factors: compared with the control group,the relative expressions of IL-1β?IL-6?TNF-α in the model group were significantly increased;Compared with the model group,the relative expressions of IL-1β?IL-6 ?TNF-α in Huayu Qutan formula drug-contained serum group contained were significantly decreased;Compared with the model group,the relative m RNA expressions of IL-1β?IL-6?TNF-α in dimethyl malonate group increased significantly;Compared with dimethyl malonate group,the relative expressions of IL-1β?IL-6?TNF-α in Huayu Qutan formula drug-contained serum intervention dimethyl malonate group decreased significantly;The differences were statistically significant(P<0.01).Experiment 3:1.Protein and m RNA expression of glycometabolism reprogramming PPP activation: the expressions of G6PD?p38MAPK?NF-κB in the model group were significantly higher than that in the control group;Compared with the model group,the expressions of G6 PD ?p38MAPK ? NF-κB in Huayu Qutan formula drug-contained serum group decreased;Compared with the model group,the expressions of G6PD?p38MAPK?NF-κB in RRX-001 group decreased significantly;Compared with RRX-001 group,the relative m RNA expressions of G6PD?p38MAPK?NF-κB in the Huayu Qutan formula drug-containing serum intervention RRX-001 group decreased significantly;The differences were statistically significant(P<0.05 or P<0.01).2.Protein and m RNA expression of glycometabolism reprogramming PPP activation inflammatory factors: compared with the control group,the expressions of IL-1β?IL-6?TNF-αin the model group were significantly increased;Compared with the model group,the expressions of IL-1β?IL-6?TNF-α in the Huayu Qutan formula drug-contained serum group were significantly decreased;Compared with the model group,the expressions of IL-1β?IL-6?TNF-α in RRX-001 group were significantly decreased;Compared with RRX-001 group,the relative expressions of IL-1β?IL-6?TNF-α in the Huayu Qutan formula drug-contained serum intervention RRX-001 group decreased significantly;The differences were statistically significant(P<0.05 or P<0.01).Conclusion:1.Huayu Qutan formula can help fight against AS by correcting blood fat disorder,intervening in inflammatory reaction and reducing the formation of aortic plaque.2.Huayu Qutan formula can help fight against AS by interfering with the occurrence and development of AS by inflammatory reactions.Its mechanism may be related to correct the reset of tricarboxylic acid cycle and inhibit the activation of pentose phosphate pathway. |
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