| Objective:In vivo experiments,we want to clarify the role of Notoginsenoside R1(NR1)in improving Atherosclerosis(AS)by inducing autophagy,regulating lipid metabolism,mediating oxidative stress,inhibiting inflammatory response to inhibiting Atherosclerosis.In addition,we investigate the mechanism of NR1 in reducing inflammatory response was explored through autophagy as a breakthrough point to induce phenotypic transformation of macrophages in vitro.Methods:1.Theoretical part : The paper reviewed the research progress on autophagy as a cellular mechanism that plays a protective role in atherosclerosis.The correlation between autophagy and phenotypic transformation of macrophages in the process of regulating inflammatory response was analyzed in depth.And combined with the pharmacological mechanisms of NR1 on cardiovascular and cerebrovascular system,which provided a theoretical basis for the intervention of NR1 on AS by Autophagy to induce the transformation of macrophages from M1 type to M2 type.2.In vivo experiment:ApoE-/-mice were fed high fat diet for 12 weeks to establish AS model.Sixty ApoE-/-male mice were randomly divided into three groups:Blend group: no special administration;NR1 group: was intraperitoneally injected with NR1 25mg/kg daily;Vehicle group: was given daily intraperitoneal injection of saline of the same volume as NR1,for 8 weeks.(1)The mice were weighed;(2)Use automatic biochemical analyzer to determine:TC、TG、HDL-C、LDL-C;(3)Serum levels of MDA、SOD、GSH-Px were detected by biochemical kit;(4)ELISA was used to measure the levels of IL-6 and IL-10;(5)Use HE to detect the plaque area of aortic root in mouse;(6)Use oil red O staining to detect Lipid deposition;(7)Use PSR to measure collagen fiber;(8)Use immunohistochemistry to measure the expression of α-SMA and CD68;(9)The ultrastructure of autophagy was observed by transmission electron microscopy;(10)Use Western blot to measure the expression of p62、LC3Ⅱ/Ⅰ,the various factors and their phosphorylation of PI3k/Akt/mTOR in blood vessels.3.In vitro experiments:Cultured mouse RAw 264.7 macrophage line,cell viability was determined by CCK8 assay.The cells were randomly divided into six groups:blank group,Ang Ⅱ model group,NR1 group,rapamycin group,3-MA group,3-MA + NR1 group.LPS and IL-4 were used to induce macrophages to polarize to M1 and M2 types,and then NR1 was given to treat cells to further study the effect of NR1 on M1 and M2 types of macrophages.(1)The ultrastructure of autophagy was observed by transmission electron microscopy;(2)Western blot was used to detect the expression of p62、LC3Ⅱ/Ⅰ,the various factors and their phosphorylation of PI3k/Akt/mTOR signaling pathway in cell supernatant;(3)The expression of CD16/32 and CD206 was detected by flow cytometry;(4)Serum levels of IL-6,Interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α)and IL-10 were measured by enzyme linked immunosorbent assay.Results:1.In vivo experiment:ApoE-/-mice fed high fat diet for 12 weeks were successfully established atherosclerosis model.(1)At the same time,the body weight of each groupt were almost indistinguishable(P>0.05).(2)Among all groups,the level of TC,TG,HDL-C and LDL-C were almost indistinguishable before treatment(P>0.05).The level of TC,TG,LDL-C decreased in each group compared with theirself after treatment(P<0.01).It was almost indistinguishable in HDL-C(P>0.05);Compared with the three groups,NR1 group was superior to the blank control group and Vehicle group in reducing serum TC,TG and LDL-C(P<0.01).(3)Oxidative stress index: Compared to blank group and vehicle group,NR1 can significantly increase the activities of SOD and GSH-Px,and reduce the content of MDA(P<0.01);(4)Inflammatory response index: Compared to blank group and vehicle group,the level of IL-6 was decreased and IL-10 was increased in NR1group(P<0.01);(5)HE staining:The AS plaque area in the aortic root decreased in NR1 group which compared to blank and vehicle group(P<0.01).(6)Oil red O staining:The lipid accumulation decreased in NR1 group which compared to blank and vehicle group(P<0.01).(7)PSR staining:The collagen fibers of elastic membrane were obviously thickened in NR1 group which compared to blank and vehicle group(P<0.01).(8)Immunofluorescence assay:The infiltration of macrophages decreased and the content of SMC increased in NR1 group which compared to blank and vehicle group.(9)Transmission electron microscopy showed that autophagosomes were increased in NR1 group;(10)Western blot method: NR1 can obviously increase LC3 Ⅱ / Ⅰ levels,inhibit P62 protein levels;The expression levels of p-Akt and p-mTOR were significantly decreased in NR1 group,but there was no significant difference of total Akt and mTOR.2.In vitro experiments:(1)The effect of NR1 on autophagy and macrophage phenotypic conversion:(1)CCK8 results: The cell survival rate was the best when cultured at 50 u M NR1 for 24h;(2)Transmission electron microscopy: Autophagosomes were increased in the NR1 group and the rapamycin group,but the phenomenon of autophagy was not obvious in the other groups.(3)Western blot : compared with the blank group,model group could significantly decreases the ratio of LC3Ⅱ/Ⅰ and increase the level of p62;compared with model group,NR1 could significantly increase the ratio of LC3Ⅱ/Ⅰ and decreases the level of p62(P<0.01),which were the same as rapamycin group(P>0.05);The rotio of LC3 Ⅱ/Ⅰwas low and the level of p62 was high in the 3-MA group,which were similar to the 3-MA+NR1 group,but compared with NR1 group,the rotio of LC3Ⅱ/Ⅰ decreased and the level of p62 increased.Compared with the model group,the rapamycin and NR1 could signifycantly inhibit the phosphorylation of Akt and Akt and mTOR(P<0.01).(4)Flow cytometry: Compared with the blank group,the expression of CD16/32 was significantly increased in the model group(P<0.01),but the expression of CD206 was no significant difference between the two groups(P>0.05);Compared with the model group,the expression of CD16/32 was significantly reduced in the NR1 group(P<0.01),the expression of CD206 was significantly increased(P<0.01),there was no statistical difference between the NR1 group and the rapamycin group(P>0.05);The expression of CD16/32 and CD206 in the 3-MA group was similar to that in the model group(P>0.05);When co-treated with 3-MA and NR1,the results were similar to those in the 3-MA alone treatment group,while the expression of CD16/32 was significantly increased and the expression of CD206 was significantly decreased compared with the NR1 group(P<0.01).(5)ELISA method: Compared with the blank group,the levels of IL-6,IFN-γ and TNF-α in model group were significantly increased(P<0.01),and the levels of IL-10 was no significant difference(P>0.05);Compared with the model group,the levels of IL-6,IFN-γ and TNF-α in NR1 group were significantly decreased(P<0.01),the levels of IL-10 was increased(P<0.01),and the results were similar to those of the rapamycin group(P>0.05);The levels of inflammatory factors in the 3-MA group were similar to those in the model group.When co-treated with 3-MA and NR1,the results were similar to those in the 3-MA alone treatment group,while the levels of IL-6,IFN-γ and TNF-α were significantly increased and the levels of IL-10 were decreased compared with the NR1 group.(2)The effect of NR1 on M1 and M2: we used LPS and IL-4 to construct M1 and M2 in vitro for experiment.(1)Flow cytometry: After LPS-induced macrophages,the expression of CD16/32 was high,while CD206 was low,presenting M1.After NR1 intervention,the expression of CD16/32 was decreased and CD206 was increased(P<0.01);The expression of CD206 was high and CD16/32 was low after IL-4induction,presenting M2.After NR1 administration,the expression of CD206 and CD16/32 had no significant changes(P>0.05);(2)ELISA: The levels of IL-6,IFN-γ and TNF-α in M1 were high,which increased after NR1 intervention,while the levels of IL-10 increased(P<0.01);The levels of IL-6,IFN-γ and TNF-α were low,while IL-10 was high,The levels of all factors did not change significantly after NR1 intervention(P>0.05).(3)Western blot: NR1 could significantly increase the ratio of LC3 Ⅱ/Ⅰ and decreases the level of p62 in M1(P<0.01);However,there were no effect in M2(P>0.05).(4)Transmission electron microscopy: The autophagy phenomenon was obvious in the LPS+NR1 group.Conclusion:1.NR1 has a definite effect on anti-AS plaque in Apoe-/-mice.It can reduce plaque of atherosclerosis area,inhibit lipid plaques,also can make the collagen fibers of elastic membrane thickening,reduce the infiltration of macrophages,increase the content of SMC to to promote atherosclerotic plaque tends to stable.2.The anti-AS effect of NR1 is related to regulating lipid metabolism,mediating oxidative stress and inhibiting inflammatory response.3.NR1 has the ability to improve the level of autophagy,and mainly through promoting autophagy of M1 to regulate the transformation of macrophages from M1 to M2 in the process of anti-AS.4.The mechanism of NR1 promoting autophagy regulation of macrophage phenotype transformation may be related to selective inhibition of PI3 K /Akt/mTOR. |
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