| Objective:To study the effect mechanism of Luhuang granule on atherosclerosis based on NNMT regulating SIRT6-PCSK9 pathway.Methods:48 8-week-old apoE-/- and 12 wild-type C57BL/6J male mice were taken,and wild-type C57BL/6J mice were given ordinary feed as a normal control group.48 apoE-/- were randomly divided into 5 groups:high-fat diet+distilled water blank control group,high-fat diet+statin positive control group,high-fat diet+low-dose Luhuang granule treatment group,The high-fat diet+ medium-dose Luhuang granule treatment group and the high-fat diet+high-dose Luhuang granule treatment group were treated with the above-mentioned treatment for 12 weeks.Full-automatic biochemical analyzer to detect mouse serum LDL-C;ELISA method to detect mouse serum me-NAM and PCSK9 levels;HE staining to detect intima-media thickness;oil red O staining to detect aortic plaque load;immunohistochemical method Macrophage CD68 in plaque;colorimetric determination of liver NAD+level;high performance liquid chromatography-ultraviolet detection（HPLC-UV）to detect the content of me-NAM in liver tissue;qRT-PCR（Quantitative Real-time Polymerase chain reaction（qRT-PCR）method for detection of Nicotinamide-N-methyltransferase（NNMT）and NAD+dependent histone deacetylase（NAD-dependent deacetylase sirtuin）in liver tissue-6,SIRT6）,proprotein convertase subtilisin kexin 9（PCSK9）and low density lipoprotein receptor（LDLR）gene expression levels;Western Blot quantifies NNMT and SIRT6,respectively,PCSK9,LDLR and histone H3K9 and H3K56 protein levels.Results:（1）Serum LDL-C content in mice:The level of serum LDL-C in the model group was higher than that in the normal control group（P<0.05）,and the level of serum LDL-C in the statin group was lower than that in the model group（P<0.05）.The level of LDL-C in the low-,medium-,and high-dose groups after decoction administration for 12 weeks was lower than that in the model group（P<0.05）.（2）Serum me-NAM content:The serum me-NAM content of the normal control group was lower than that of the model group（P<0.05）,and the serum me-NAM content of the statin group was significantly higher than that of the model group（P<0.05）The lu-granule high-dose group had a lower me-NAM content than the model group（P<0.05）.（3）Serum PCSK9 content:The PCSK9 content of the model group was significantly higher than that of the normal control group（P<0.05）.Compared with the model group,the PCSK9 content of the statin group and high-dose group decreased significantly（P<0.05）.Reduced（P<0.05）.（4）Inner media thickness of mouse aorta:model group>Luhuang granule low dose group>Luhuang granule medium dose group>statin group>Luhuang granule high dose group>normal control group.（5）Oil red O staining to detect aortic plaque load:the plaque load of the normal control group was significantly lower than that of the model group（P<0.05）,the aortic plaque load of the statin group and the high and low dose groups of Luhuang granules were less than the model Group（P<0.05）.（6）Immunohistochemical detection of macrophage CD68:model group（M）>Luhuang granule low-dose group（D）>Luhuang granule middle-dose group（Z）>Luhuang granule high-dose group（G）>statin group（T）>Normal control group（Y）.（7）HE staining to observe liver morphological changes:liver fat changes from heavy to light:model group>Luhuang granule low dose group>Luhuang granule middle dose group>statin group>Luhuang granule high dose group>normal control group.（8）HPLC-UV detection of liver NNMT activity（me-NAM content）:the me-NAM content of the model group was significantly higher than that of the normal control group（P<0.05）;the statin group,Luhuang granule high,medium and low dose groups Compared with the model group,the content of me-NAM decreased significantly（P<0.05）.（9）Determination of liver NAD+level by color rendering method:The results show that the NAD+content of the model group is significantly higher than that of the normal control group（P<0.05）.In the model group（P<0.05）.（10）Gene expression levels of NNMT,SIRT6,PCSK9,LDLR:the normal control group LDLR and SIRT6 gene expression levels were significantly higher than the model group（P<0.05）,the statin group LDLR,SIRT6 gene expression levels were greater than the model group（P<0.05）,the expression levels of LDLR and SIRT6 in the low,medium and high dose groups of Luhuang Granule were higher than those in the model group（P<0.05）;The NNMT and PCSK9 gene expression levels of the normal control group were lower than those of the model group（P<0.05）,the NNMT and PCSK9 gene expression levels of the statin group were lower than those of the model group（P<0.05）,and the medium and high dose groups of Luhuang granules had NNMT,PCSK9 gene expression level lower than the model group（P<0.05）.（11）NNMT,SIRT6,PCSK9,LDLR and histone H3K9 and H3K56 protein levels:the protein expression of PCSK9,NNMT,H3K9,H3K56 in the model group was significantly higher than that of the normal control group（P<0.05）,the expression of LDLR,SIRT6 The model group was significantly lower than the normal control group（P<0.05）,the protein expression of PCSK9,NNMT,H3K9,H3K56 in the statin group was significantly lower than the model group（P<0.05）,and the protein expression of LDLR and SIRT6 was significantly higher than the model group（P<0.05）,the PCSK9,NNMT,H3K9,H3K56 protein levels of the Luhuang granules were significantly lower than the model group（P<0.05）,while the LDLR and SIRT6 protein expression levels were only in the high dose of Luhuang granule The difference between the group and the model group was statistically significant（P<0.05）.Conclusion:Luhuang granule may affect the process of NAD+by regulating NNMT,reducing the activity of NNMT,further down-regulating NAD+,and increasing the activity of SIRT6,thereby inhibiting the expression of PCSK9,further suppressing serum LDL-C content in the body,and inhibiting atherosclerotic development. |
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