Objective

2

To investigate the possible mechanism of action and combination of medicinals of

Zhuyu Pill

（茱萸丸） in the treatment of atherosclerosis.

Methods

2

Thirteen normal C57BL/6J mice were used as blank group， and 40 ApoE

-/-

mice of the same strain were randomly divided into model group， Huanglian （

Coptis chinensis Franch.

） group， Wuzhuyu （

Tetradium ruticarpum

） group， and

Zhuyu Pill

group， with 10 mice in each group. Except the blank group， the other groups were fed with high-fat diet for modeling， and the total modeling cycle was 12 weeks. After modelling， Huanglian group was given Huanglian solution 143.34 mg/（kg·d）， Wuzhuyu group with Wuzhuyu solution 301.78 mg/（kg·d），

Zhuyu Pill

group with 261.07 mg/（kg·d） of

Zhuyu Pill

solution， blank group and model group were given 0.3ml of normal saline. Mice in all groups were gavaged for 12 weeks. The levels of total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C） and fasting blood glucose were measured by biochemical method； fasting insulin in serum was detected by enzyme-linked immunosorbent assay （ELISA） and insulin resistance index was calculated； HE staining was used to observe the pathological and morphological changes of aortic tissue， liver and epididymal fat in mice， and oil red O staining was used to observe the lipid deposition of aortic tissue in mice， and the percentage of positive area of aortic HE staining， the percentage of positive area of aortic oil red O staining， the non-alcoholic fatty liver activity score （NAS） score， and the cross-sectional area of epididymal fat were calculated； serum levels of interleukin 1β （IL-1β） and interleukin 18 （IL-18） were detected by ELISA； protein expression of nucleotide-binding oligomerisation structural domain-like receptor family pyridoxal structural domain protein 3 （NLRP3） in aortic tissues was detected by Western blot； and immunofluorescence was used to detect the levels of apoptosis-associated granulocyte-like protein （ASC）， which contains the CARD structural domain （ASC） and cysteine-containing aspartic protease 1 （Caspase-1） protein expression； and real-time fluorescence PCR was used to detect NLRP3， ASC， Caspase-1， IL-1β， and IL-18 mRNA expression in mouse aortic tissues.

Results

2

Compared with blank group， serum TC， TG and LDL-C were increased， insulin was decreased， fasting blood glucose and insulin resistance index were increased in model group （

P

＜0.01）. Aortic plaque area increased， liver lipid deposition increased， epididymal fat cells volume increased， liver NAS score increased， and epididymal fat cross-sectional area increased. Serum IL-1β and IL-18 increased， and the expression levels of NLRP3， ASC， Caspase-1 protein and mRNA in aortic tissue increased （

P

＜0.01）. Compared with model group， serum TC， TG and LDL-C in Huanglian group， Wuzhuyu group and

Zhuyu Pill

group decreased， fasting insulin increased， fasting blood glucose and insulin resistance index decreased （

P

＜0.01）； aortic plaque area decreased significantly， liver lipid deposition decreased， liver NAS score decreased， epididymal fat cell volume decreased， epididymal fat cross section area decreased， serum IL-1β and IL-18 were decreased， the expression levels of NLRP3， ASC， Caspase-1 protein and mRNA in aortic tissue were decreased （

P

＜0.01）， and the improvement of all indexes in the

Zhuyu Pill

group was better than that in Huanglian and Wuzhuyu groups （

P

＜0.01）.

Conclusion

2

Zhuyu Pill

has a good therapeutic effect on atherosclerosis in mice， and the combination of Huanglian and Wuzhuyu has a synergistic effect， the mechanism of which may be related to regulation of the NLRP3/ASC/Caspase-1 aortic signaling pathway， and thus reducing the vascular inflammatory response.