| Purpose:Experiments show that PI3K/Akt signaling pathway can activate autophagy of macrophages,reduce the excessive secretion of inflammatory factors and inhibit AS process.A number of studies have found that berberine(Ber),an effective extract of some traditional Chinese medicine,has a significant anti-AS effect.However,whether Ber can promote autophagy of macrophages,reduce inflammation and increase the stability of vulnerable plaques by regulating PI3K/Akt pathway,and thus play an anti-AS effect has not been clarified.This study focuses on the PI3K/Akt pathway,macrophage autophagy and inflammatory response,and discusses the anti-AS effect of Ber and its intervention mechanism,in order to provide new theoretical support and new research ideas for the prevention and treatment of AS by traditional Chinese medicine.Method:A total of 10 male mice were fed ordinary diet AS a blank control group,and male apolipoprotein E deficiency(Apo E-/-)mice were fed high-fat diet to create AS animal model,and were randomly divided into 7 groups with 10 mice per group.They were(2)AS model group,(3)LY294002 group,(4)triciribine group,(5)simvastatin group,(6)Ber low-dose group,(7)Ber medium-dose group,(8)Ber high-dose group.(3)The group was given Ber 7.5 mg·kg-1·d-1,followed by PI3 K inhibitor LY294002(2 mg LY294002 dissolved in DMSO plus 0.25 m L PBS)by intraperitoneal injection for 8 weeks.Group 4 was given Ber 7.5 mg·kg-1·d-1 and then given Akt inhibitor triciribine(1 mg·kg-1·d-1)for 8 weeks.Group 5 was given simvastatin solution(5mg/kg·d-1)for 8 weeks.(6),(7)and(8)groups were given Ber 2.5,5 and 7.5 mg·kg-1·d-1,respectively,and were intragastrically intragastrically for 8 weeks.Groups(1)and(2)were given equal dose of normal saline,once a day,and their body mass was measured once each time.The experimental cycle was 16 weeks.AS plaque morphological changes were observed by oil red O and HE staining,and lipid accumulation and plaque area percentage in aorta of mice in each group were compared.The content of macrophage autophagosomes in plaque was observed by transmission electron microscopy.Western blot was used to detect the expression levels of p-PI3 K,p-Akt,Beclin1,LC3-II,IL-6,TNF-α and hs-CRP in aorta.The levels of serum inflammatory markers IL-6,TNF-α and hs-CRP were detected by ELISA(enzyme-linked immunosorbent assay),and the distribution of PI3 K and Akt in AS plaques were detected by immunohistochemical staining.Results:1.Morphological test results of mouse aorta AS plaques: The intima of mouse aorta tissue in blank control group was smooth and continuous,the results were complete,and no lipid deposition and AS plaques were found in the intima.In the AS model group,the structure of the intima of mouse aorta tissue was not complete,and a large number of inflammatory cells were infiltrated,lipid accumulation was observed,and AS plaques were formed.Compared with the AS model group,the AS lesions in LY294002 group,triciribine group,simvastatin group,Ber medium dose group and Ber high dose group were improved to different degrees,and the AS lesions in the Ber medium dose group were similar to those in the AS model group.(1)Oil red O staining showed no significant lipid accumulation in the aorta vessels of mice in the control group.Compared with the control group,lipid accumulation in aorta vascular tissue of mice in AS model group was significantly increased(P < 0.05).Compared with AS model group,lipid accumulation in aorta vascular tissue of mice in simvastatin group,Ber medium dose group and Ber high dose group was significantly decreased(P < 0.05),but there was no significant difference in lipid accumulation in aorta vascular tissue of mice in Ber low dose group(P>0.05).Compared with Ber high dose,aortic lipid accumulation in LY294002 group and triciribine group was significantly decreased(P < 0.05).(2)HE staining showed no AS lesion in the aorta of blank control group.Compared with the control group,the percentage of AS plaque area in aortic vascular tissue of mice in AS model group was significantly increased(P < 0.05).Compared with AS model group,the percentage of AS plaque area in simvastatin group,Ber medium-dose group and Ber high-dose group was significantly decreased(P < 0.05),but there was no significant difference in the percentage of AS plaque area in Ber low-dose group(P>0.05).Compared with Ber high dose,the percentage of aortic AS plaque area in LY294002 group and triciribine group was significantly decreased(P <0.05).2.Immunohistochemical staining results: PI3 K and Akt positive expression particles were observed in the brownish yellow area of each section under light microscope,and no AS plaque was found in the aorta tissue of blank control group.Compared with the control group,the positive expression particles of PI3 K and Akt in AS plaques of mice in AS model group were significantly increased(P < 0.05).Compared with AS model group,the positive expression particles of PI3 K and Akt in AS plaques of mice in simvastatin group,Ber medium-dose group and Ber high-dose group were significantly decreased(P < 0.05),while the positive expression particles of PI3 K and Akt in AS plaques of mice in Ber low-dose group were not significantly different(P>0.05).Compared with Ber high dose,the positive expression particles of PI3 K and Akt in AS plaques in LY294002 group and triciribine group were significantly reduced(P < 0.05).3.Observation of macrophage autophagosomes in AS plaques under transmission electron microscopy: A small number of autophagosomes were observed in aortic macrophages of mice in the control group.Compared with the control group,the content of autophagosomes in AS plaques in AS model group was significantly decreased(P < 0.05).Compared with AS model group,the content of autophagosomes in AS plaques of mice in simvastatin group,Ber medium-dose group and Ber high-dose group was significantly increased(P < 0.05),while the content of autophagosomes in AS plaques of mice in Ber low-dose group was not significantly different(P >0.05).Compared with Ber high dose,the content of autophagosome in AS plaques in LY294002 group and triciribine group was significantly increased(P < 0.05).4.Enzyme-linked immunosorbent assay(ELISA)results: Compared with the control group,the contents of inflammatory factors(IL-6,TNF-α,hs-CRP)in AS model group were significantly up-regulated(P < 0.05);Compared with AS model group,the contents of inflammatory factors(IL-6,TNF-α,hs-CRP)in simvastatin group,Ber medium-dose group and Ber high-dose group were significantly decreased(P < 0.05),while the contents of inflammatory factors(IL-6,TNF-α,hs-CRP)in Ber low-dose group were not significantly different(P>0.05).Compared with Ber high dose,the contents of inflammatory cytokines(IL-6,TNF-α and hs-CRP)in LY294002 group and triciribine group were significantly decreased(P < 0.05).5.Western blot test results:(1)The expression levels of P-PI3 K and P-Akt in aortic vascular tissue: compared with the control group,the expressions of P-PI3 K and P-Akt in AS model group were significantly increased(P < 0.05);Compared with AS model group,the protein expressions of P-PI3 K and PAkt in simvastatin group,Ber medium-dose group and Ber high-dose group were significantly decreased(P < 0.05),while the protein expressions of P-PI3 K and P-Akt in Ber low-dose group were not significantly different(P>0.05).Compared with Ber high dose,the protein expressions of P-PI3 K and P-Akt in LY294002 group and triciribine group were significantly decreased(P <0.05).(2)Expression levels of inflammatory cytokines IL-6,TNF-α and hs-CRP in aorta: There were small amounts of inflammatory cytokines IL-6,TNF-α and hs-CRP in tissue samples of mice in control group.Compared with the control group,the expressions of inflammatory cytokines IL-6,TNF-α and hs-CRP in the tissue samples of mice in AS model group were significantly upregulated(P < 0.05).Compared with AS model group,the expressions of inflammatory cytokines IL-6,TNF-α and hs-CRP in mouse tissue samples of simvastatin group,Ber medium dose group and Ber high dose group were significantly down-regulated(P < 0.05).There were no significant differences in the expressions of inflammatory cytokines IL-6,TNF-α and hs-CRP in Ber lowdose group(P>0.05).Compared with Ber high dose,the expressions of inflammatory cytokines IL-6,TNF-α and hs-CRP in mouse tissue samples of LY294002 group and triciribine group were significantly down-regulated(P < 0.05).(3)Expression levels of autophagy markers Beclin1 and LC3-II proteins: small amounts of Beclin1 and LC3-II proteins were found in aorta tissues of mice in the control group.Compared with the control group,Beclin1 and LC3-II protein expressions in aorta tissues of mice in AS model group were significantly decreased(P < 0.05).Compared with AS model group,Beclin1 and LC3-II protein expressions in simvastatin group,Ber medium-dose group and Ber high-dose group were significantly increased(P < 0.05),while Beclin1 and LC3-II protein expressions in Ber low-dose group were not significantly different(P>0.05).Compared with Ber high dose,Beclin1 and LC3-II protein expressions in aorta tissue of mice in LY294002 and triciribine groups were significantly increased(P < 0.05).Conclusion:Berberine has a significant anti-AS effect,and its mechanism may be related to its regulation of PI3K/Akt signaling pathway,promoting autophagy of macrophages and reducing inflammatory response. |
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