| Objective: To study the effects of Ziziphora clinopodioides Lam.on TLR4/NF-?b and Wnt/?-Catenin signaling pathways and their downstream factors in ApoE-/-atherosclerotic mice,and to explore the mechanism of Ziziphora clinopodioides Lam.in preventing and treating inflammation of atherosclerosis.Methods:(1)60 8-week-old ApoE-/-mice and 12 C57 mice of the same strain were fed with high-fat diet for 94 days to establish atherosclerosis model.After feeding all mice for 1 week,ApoE-/-mice were randomly divided into model group [0.4 mL/10 g,distilled water],Statin group [3mg/(kg.d),statin],Tianxiangdan group [2.7 g/(kg.d),Tianxiangdan granule],Ziziphora clinopodioides Lam.high dose group [1.8 g/(kg.d),water extract],Ziziphora clinopodioides Lam.low dose group [0.9 g/(kg.d),water extract],C57 were blank group [0.4 mL/10 g,distilled water],10 mice in each group,the weight changes of mice were recorded weekly,and the dosage of C57 was adjusted.After 12 weeks of intragastric administration,all mice were fasting for 12 hours without water,Tissue specimens taken after anesthesia.(2)The changes of body weight and serum lipids in mice were observed.(3)The pathological changes of thoracic aorta in mice were observed by hematoxylin eosin(HE)staining.(4)The serum levels of TNF-?,IL-1?,IL-6,IL-10,ICAM-1 and MMP-9 were detected by ELISA.(5)The mRNA expression of TLR4,NF-?b,TNF-?,IL-1?,IL-6 and IL-10 in myocardium were detected by RT-qPCR.(6)Western Blot was used to detect the expression of GAPDH,TLR4,p-NF-?bp65,Wnt1,Wnt3 a and ?-Catenin in abdominal aorta.(7)The pathological changes of ascending aorta and aortic arch were observed by immunohistochemistry(IHC).The expressions of TLR4,p-NF-?bp65,Wnt1,Wnt3 a and?-Catenin were detected.Results:(1)Weight and blood lipid of mice: The weight of micein each group showed an upward trend.Compared with model group,statin group,Tianxiangdan group,Ziziphora clinopodioides Lam.high-dose group and Ziziphora clinopodioides Lam.low-dose group had a certain effect of reducing lipid and reducing weight gain of mice(P <0.01 or P <0.05).(2)ELISA results showed that the levels of TNF-?,IL-1?,IL-6,ICAM-1 and MMP-9 in the model group were significantly higher than those in the blank group(P <0.01),while the levels of IL-10 were significantly lower than those in the blank group(P <0.01);the levels of TNF-?,IL-1?,IL-6,ICAM-1 and MMP-9 in the statin group,Tianxiangdan group and Ziziphora clinopodioides Lam.high dose group were lower than those in the model group(P <0.01).The levels of serum ICAM-1 and MMP-9 in the low dose group of Ziziphora clinopodioides Lam.were lower than those in the model group(P <0.01 or P <0.05),and the levels of IL-10 were higher than those in the model group(P <0.05).The levels of TNF-?,IL-6 and IL-1? in the low dose group of Ziziphora clinopodioides Lam.were not significantly different from those in the model group(P >0.05).(3)RT-qPCR results showed that the expression levels of TLR4,NF-?b,TNF-?,IL-1? and IL-6 in the model group were higher than those in the blank group(P <0.01 or P <0.05),while the expression levels of IL-10 were lower than those in the blank group(P<0.01);the expression levels of TLR4,NF-?b,TNF-?,IL-1?and IL-6 in the myocardium of statin group,Tianxiangdan group and Ziziphora clinopodioides Lam.high dose group were higher than those in the blank group(P <0.01).Compared with the model group,the expression level of IL-10 was significantly higher(P<0.01 or P <0.05),and the expression level of IL-10 was significantly higher(P <0.01 or P <0.05);the expression levels of NF-?b and IL-1? in myocardium of Ziziphora clinopodioides Lam.low-dose group were lower than those of model group(P <0.01 or P<0.05),and the expression levels of TNF-?,IL-6,IL-10 and TLR4 were significantly lower than those of model group(P <0.01 or P <0.05).There was no significant difference(P >0.05).(4)Western Blot results showed that the expression of TLR4,p-NF-?bp65,Wnt1,Wnt3 a and ?-Catenin protein in model group was significantly higher than that in blank group(P <0.01);compared with model group,the expression levels of TLR4,p-NF-?bp65,Wnt1,Wnt3 a and ?-Catenin protein in arteries of statin group,Tianxiangdan group and Ziziphora clinopodioides Lam.high dose group were significantly lower(P <0.01 or P<0.05).The levels of TLR4,p-NF-?bp65 and Wnt1 in the low-dose group of Ziziphora clinopodioides Lam.were significantly higher than those in the model group(P <0.01 or P<0.05).The levels of Wnt3 a and ?-Catenin protein in the low-dose group of Ziziphora clinopodioides Lam.were not significantly different from those in the modelgroup(P >0.05).(5)IHC results showed that the expression of TLR4,p-NF-?bp65,Wnt1,Wnt3 a and ?-Catenin protein in model group was up-regulated compared with blank group(P <0.01);compared with model group,the expression levels of TLR4,p-NF-?bp65,Wnt1,Wnt3 a and ?-Catenin protein in artery of statin group,Tianxiangdan group and Ziziphora clinopodioides Lam.high dose group were down-regulated(P <0.01).The levels of TLR4 and Wnt1 in the low dose group were significantly higher than those in the model group(P<0.01).The levels of p-NF-?bp65,Wnt3 a and ?-Catenin in the low dose group of Ziziphora clinopodioides Lam.were not significantly different from those in the model group(P >0.05).Conclusion: Ziziphora clinopodioides Lam.can effectively prevent and treat atherosclerosis.Its mechanism may be related to inhibiting the protein levels and downstream factors related to TLR4/NF-?b and Wnt/?-Catenin signaling pathways,reducing inflam-mation,and suggesting that it may be dose-dependent. |
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