| Cardiovascular disease caused by atherosclerosis seriously affects human life and health. The existing drugs for resisting atherosclerosis are mainly purpose to depress blood fat, for example, fibrate antihyperlipidemic drugs, statins, bile acid isolation agent, nicotinic acid and so on. While fibrate, bile acid isolation agent and nicotinic acid not only can lead to severe gastrointestinal discomfort, liver and kidney function damage, but also are selective low and easy to tolerate, so they are used fewer now. Currently statins are the most widely used drug for treating atherosclerosis, which have some side effects such as muscle pain and liver toxicity. Morbidity of cardiovascular disease caused by Atherosclerosis remains high, suggesting besides hyperlipidemia, there may still have other risk factors.With the further research on the pathogenesis of atherosclerosis, it has been widely recognized the inflammatory immune factors has a very important role in the process of initiation and progression of atherosclerosis. Our early studies have confirmed that inflammation stimulation can significantly increase the formation of atherosclerotic plaque in a variety of animal model, and anti-inflammatory therapy helps reducing plaque formation. Interleukins play important roles in the chronic vascular inflammation in atherosclerosis. For example, IL-1 can promote early plaque formation and leukocyte adhesion to endothelial cells, IL-18 raise the expression of macrophage and endothelial cell adhesion molecule, and vascular cell adhesion molecule. Studies have found that after NLRP3 inflammasome were activated by a variety of incentive, NLRP3 can cut precursor of IL-1β and IL-18, and prompt them matured and released into the extracellular so that cause inflammation. And compared with other members of the family of the character, NLRP3 inflammasome is the main cause to prompt IL-1β mature and release. So restraining the activity of NLRP3 inflammasome is likely to play an important role in reducing the formation of atherosclerosis. In conclusion, From adjusting blood fat and reducing the local inflammatory response to take intervention measures at the same time probably will be a new strategy for preventing and treating atherosclerosis.Our lab has found PNS can cause significant resistance to atherosclerosis by adjusting blood fat and anti-inflammatory effects. Our pre-experiments have showed that compound K may be the main active ingredient of PNS for resisting atherosclerosis. In addition, Our studies have found that function of PNS to reduce the action of the foam cell formation is related to the increased expression of ABCA1 which is LXRα downstream target genes. Also, PNS can reduce atherosclerotic plaque formation in rats by inducing LXRα expression. But the effects and mechanisms of PNS on atherosclerosis are still unknown.So this topic mainly discuss CK effects on atherosclerosis formation, and explore its mechanism from Reverse cholesterol transport process and NLRP3 inflammasome. Overall, this finding may provide experimental foundation for CK as a new drug for preventing and treating atherosclerosis.Methods:1. Mice and treatments: After adaptive breeding in SPF room for a week, 42 male C57BL/6 apo E-/- mice(8-week old) were randomly divided into seven groups(n=6/each group): mice of control group were administrated with 20 ml/kg normal saline and fed a normal diet; mice of model group were treated with 20 ml/kg normal saline and fed a Western diet(normal diet containing 0.5% cholesterol and 5% lard); three groups were treated with CK at doses of 1, 3 and 9 mg/kg respectively, together with Western diet; GGPP group was administrated with CK(3 mg/kg) plus 9 mg/kg GGPP based on Western diet; one group as positive control was treated with Atorvastatin(2.6 mg/kg), together with Western diet. All of the treatments were given by intraperitoneal injection everyday and lasted 12 weeks. Both water and food were available ad libitum. During the treatments, changes in the body weight of mice were monitored once a week. Tissue samples were obtained after 12 weeks.2. The aortic sinus were sectioned after fixation and embedding, then stained with hematoxylin-eosin(H&E) for histopathological analysis. Frozen pathological section was used to deal with liver tissue for Oil-Red O staining for the measurement of hepatic lipid accumulation. Image analysis was carried out with Nis-Elements BR 3.2 software.3. 500μL blood of each mouse was sampled by removing eyeball, centrifuged in 3000 rpm for 10 min at 4℃. The levels of serum lipids were tested by an AU-2700 automatic biochemical analyzer.4. The levels of serum typically cytokines including tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) was determined by Millpex catalog ID according to the manufacturer’s instructions.5. Proteins from mice artery, small intestine and liver were extracted. Then proteins expression including LXRα, ABCA1, ABCG1 in artery, ABCG5, ABCG8 in small intestine and SREBP-1c in liver were detected.6. Total proteins of C57 BL / 6 mice abdominal cavity macrophage derived foam cells were extracted. The expression of ABCA1 protein was tested.7. Proteins from artery and liver tissue were extracted. The expression of NLRP3, Caspase-1 and IL-1β were tested.8. C57 BL / 6 mice abdominal cavity macrophage derived foam cells were cultivated and undertook according to the following groups: control group, ox-LDL(100μg/ml) model group, low-dose group of CK(ox-LDL + 3.3μM CK), mid-dose group of CK(ox-LDL +10μM CK), high-dose group of CK(ox-LDL +30μM CK), group of CK and GGPP(ox-LDL +10μM CK+10μM GGPP), GW3965 group(ox-LDL +10μM GW3965). Cholesterol content in foam cells was detected after cultivating for 24 h.9. Logarithmic growth phase cells were induced to be macrophage by PMA for 24 h, and were disposed as follow: control group, cholesterol(1μg/L) model group, high-does GW3965 group(cholesterol + 30μM GW3965), mid-dose GW3965 group(cholesterol + 10μM GW3965), low-dose GW3965 group(cholesterol + 10μM GW3965), group of GW3965 and GGPP(10μM GW3965+10μM GGPP), GGPP group(10μM GGPP), ABCA1 antibody group(10μM GW3965+1:200 ABCA1antibody), apo E group(10μM GW3965+1:200 apo E antibody). In addition, 100ng/ml LPS as model group for testing function of LPS, other groups were given the same drugs as cholesterol experiment. Total RNA and total protein were extracted and detected.10. C57 BL / 6 mice abdominal cavity macrophage derived foam cells were cultivated. Grouping and dosing method are the same as above mentioned. The expression of NLRP3, Caspase-1 and IL-1β were tested.Results1. After aorta of apo E-/- mice were selectioned and stained by HE, Quantitative image analyses of microscopic aortas slides revealed noticeable lesion in model group(15.05±1.97%). The atherosclerotic plaque was reduced to 6.58±1.09% and 2.16±0.62% by 3 mg/kg and 9 mg/kg respectively, indicating that CK attenuated the formation of atherosclerotic plaque in apo E-/- mice dose-dependently.2. Abundant oil-red O staining lipid depositions in the livers were observed in apo E-/-mice. The area ratio of positive staining is(32.00±4.91%). Significant decrease of the positive staining area ratio was found in CK 3 mg/kg(11.85±2.40%) and 9 mg/kg(4.07±0.76%) groups, as well as in atorvastatin(4.39±1.08%) group. There was no difference of the ration of oil-red O stained area between model group and GGPP group.3. Results of foam cell showed that CK could attenuate the cholesterol deposition in foam cells dose dependently, GW3965 had the same effect, while GGPP could increase cholesterol deposition of foam cells by inhibiting the effect of CK.4. Levels of TC, TG, LDL and HDL in model group mice were noticeable increased compare with which in control group. Treatment of atorvastatin could down-regulated the serum TC and LDL significantly. There is no significant difference of TC and TG levels between model group and all of CK groups. But noticeable decrease of LDL level and increase of HDL level were found in CK 3 mg/kg and CK 9 mg/kg group, compare with model group. There was no significant change occurred after the treatment with CK 3 mg/kg together with GGPP.5. Results of protein expression in both animal and cell levels showed that CK could increase the expression level of LXRα, ABCA1 and ABCG1 dose dependently.6. Results about GW3965 experiment showed that GW3965 could dose dependently decreased m RNA level(p<0.05) and protein level(p<0.05) of NLRP3, Caspase-1, then lower the production of Cleaved IL-1β. GGPP could significantly suppress the effects of GW3965.7. Compared with control group, high-cholesterol diet in model group resulted in significant increase of serum IL-1β, IL-6 and TNF-α levels in model group. Treatment of CK(3, 9 mg/kg) could down-graduate all of the three inflammatory cytokine levels significantly. Atorvastatin could significantly reduce the levels of serum IL-1β and TNF-α, but not IL-6. Administration with additional GGPP inhibited the effects of CK 3 mg/kg-induced decline of serous cytokines.8. Results of protein expression in vivo and in vitro revealed CK could dose dependently reduce the expression of NLRP3, Caspase-1 and IL-1β, GGPP could significantly suppress the effects of CK.Conclusion1. CK can significantly attenuate the formation of atherosclerotic plaque, and can reduce lipid deposition in liver.2. Effects that CK attenuate the formation of atherosclerosis are related to promoting the RCT process and inhibiting NLRP3 inflammasome. |
PDF Full Text Request