Visfatin Involved In Atherogenesis Through The Activation Of MAPKs Pathway And The Interventive Effect Of Berberine

Background:Atherosclerosis is a serious disease which is harm to human health. The morbidity and mortality of heart and cerebrovascular disease which are caused by atherosclerosis are very high in the US and European countries. With the social and economic development, the morbidity and mortality caused by atherosclerosis in China increases year by year.The reason for the formation of AS is very complex and the pathogenesis of atherosclerosis is not yet completely clear. Chronic inflammation played a key role in the development of atherosclerosis, anti-inflammatory therapy is an important strategy of prevention and cure of atherosclerosis. Visfatin is an adipokine, clinical study found a significant rise of serum visfatin in the AS patients, and increased expression of inflammatory markers. Therefore, we hypothesized that visfatin is involved in the development of atherosclerosis.Mitogen activated protein kinases (MAPKs) signaling pathway exists in most of the cells, which are mainly p38MAPK, ERK and JNK pathway. JNK pathway and p38MAPK pathway of inflammatory cytokines and stress signal conduction, multiple types of cells and ERK main fragmentation of cell growth, differentiation and signal conduction, studies have shown that p38MAPK, ERK and JNK pathways are involved in the pathological process of the atherosclerosis. We hypothesized that visfatin pro-inflammatory mechanism may be related to regulation MAKPs signaling pathways.There is no "atherosclerosis" in traditional Chinese medicine (TCM), it is generally believed it belongs to "obstruction","stroke" and "heartburn" in TCM. Modern medicine usually give priority to with statins in the treatment of atherosclerosis, but the long-term use of statins can produce side effects such as hepatotoxicity, myopathy, neurotoxicity and gastrointestinal reaction which restrict its widespread use in clinical. Berberine, as an effective traditional Chinese medicine extraction, play a role of resistance to atherosclerosis, but if its resistance atherosclerosis mechanism is adjusting the expression of visfatin is lack of further studies. We speculated that berberine may delay the development of atherosclerosis by down-regulating the expression of visfatin, and this mechanism may be related to regulation MAKPs signaling pathways.AopE knockout (ApoE-/-) mice has become the most used atherosclerosis research of animal model because of spontaneous injury of the accumulation of foam cells and significantly higher plasma cholesterol in atherosclerosis. Vascular endothelial cell damage occurred in the early stages of atherosclerosis and the protecting of vascular endothelium is the key to prevent atherosclerosis. Human umbilical vein endothelial cells (HUVEC) are similar as arterial endothelial cells in many ways of biological characteristics, and arised widespread attention in vessel function pathological physiology and mechanism research of atherosclerosis. ApoE-/-mice were fed with a western diet with the intention of establishing atherosclerosis model. HUVEC were used to atherosclerosis cell model, studies the pro-inflammatory role of visfatin in atherosclerosis in vivo and in vitro respectively, used MAPKs signaling pathways specific inhibitors to detect the pro-inflammatory mechanism in atherosclerosis. Additional, berberine was used to determin the intervention effect and its anti-inflammation mechanism that induced by visfatin which can look for new targets for prevention of atherosclerosis by TCM.Objectives:1. To establish atherosclerosis model, investigate the visfatin expression in mice serum and aorta tissue and explore the interventive effect of berberine;2. To investigate the serum lipid, plasma interleukin-6(IL-6), tumor necrosis factor alpha (TNF-a) contents of the AS mice and explore the interventive effect of berberine;3. To investigate the plaque area quantification of mice aorta, examine the visfatin expression in the atherosclerosis plaque, and explore the interventive effect of berberine.4. To investigate whether visfatin can induce HUVEC secrete inflammatory cytokines IL-6and TNF-a, and whether its pro-inflammation is related to p38MAPK, ERK and JNK signaling pathway activation.5. To investigate the intervention effect of berberine in the process of visfatin induced HUVEC secretion the inflammatory factors IL-6and TNF-a, and whether its mechanism is related to p38MAPK, ERK and JNK signaling pathway activation.Methods:1. Animal experimentsFifty male8-week-old ApoE-/-mice were fed with a western diet and were randomly assigned into five groups:rmodel group, low-dose berberine (2.5mg/kg/d) group, medium-dose berberine (5mg/kg/d) group, high-dose berberine (7.5mg/kg/d) group and Simvastatin (2.5mg/kg/d) group, while10C57BL/6J mice on a common diet as a control group. Mice were weighed every week. The whole experiment period was12weeks.Mice were anesthetized by intra-peritoneal injection with10%chloral hydrate, following eyeball extirpation to collect their blood. The aorta was harvested and fixed in4%buffered paraformaldehyde to obtain an initial fixation.The aorta was embedded in paraffin and cross-sectioned at a5μm for Hematoxylin and eosin (H&E) staining. All images were captured with a camera and analyzed using Image Pro plus6.0to quantify the area of the atherosclerosis lesions. To identify the distribution of visfatin in atherosclerotic plaques, we measured its levels in atherosclerotic lesions by immunohistochemistry. Serum and plasma was obtained after centrifugation of the collected blood samples. Serum concentration of total cholesterol (TC), total triglycerids (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) was determined by enzymatic procedures. Levels of serum visfatin, plasma TNF-a and IL-6were analyzed by an enzyme-linked immuno-sorbent assay (ELISA) method. SPSS13.0was used for data analysis, P<0.05was considered statistically significant.2. Cell experimentsHUVEC were divided into the following groups:Visfatin concentration gradient groups:0,50,100,200μg·L-1visfatin respectively stimulate HUVEC for24h.Visfatin time gradient group:100μg·L-1visfatin stimulate HUVEC respectively for0,6,12,24and48h.Drug intervention group, which is divided into seven groups:(1) the control group;(2) the berberine group:50μmol·L-1berberine incubated for24h;(3) visfatin +berberine group:50μmol·L-1berberine beforehand to added for1h incubation before100μg·L-1visfatin stimulate HUVEC for24h;(4)visfatin group:added100μg·L-1visfatin to stimulate HUVEC for24h;(5)visfatin+PD98059group:added PD98059(final concentration of20μmol·L-1) pretreatment HUVEC for30min before100μg·L-1visfatin to stimulate HUVEC for24h;(6)visfatin+SB203580group:added SB203580(final concentration of20μmol·L-1) pretreatment HUVEC for30min before100μg·L-1visfatin to stimulate HUVEC for24h;(7)visfatin+SP600125group:added SP600125(final concentration of10μmol·L-1) pretreatment HUVEC for30min before100μg·L-1visfatin to stimulate HUVEC for24h.We detect the influence of visfatin on HUVEC proliferation and the intervention effect of berberine by MTT assay. HUVEC supernatant were collected to determin the contents of IL-6and TNF-a by ELISA and to study the role of visfatin in inducing HUVEC secrete inflammatory factors and the intervention effect of berberine. HUVEC were lysed in a lysis buffer for30min on ice. After centrifugation at12000g for15min at4℃, the supernatants were collected as total proteins in HUVEC, the protein concentrations were also determined with a BCA assay kit, western blotting was used to detect p38MAPK, ERK and JNK protein and phosphorylation protein expression, to study the mechanism in visfatin induced HUVEC inflammatory response, the relationship between three kinds of MAPKs signal pathways and the intervention effect of berberine in preventing AS. SPSS13.0was used for data analysis, P<0.05was considered statistically significant.Results:1. Mice weight measurement results showed that all the add weight value of each dosage group were statistically significant compared with model group (P=0.00). The add weight values of simvastatin group were no significant difference compared with high-dose berberine group, medium-dose berberine group and low-dose berberine group,(P>0.05). The add weight values of medium-dose berberine group there were no significant difference in the add weight values among high-dose berberine group, medium-dose berberine group and low-dose berberine group (P>0.05).2. Serum lipids measurement results showed that serum lipid levels of model group were significantly higher than control group (P<0.05), the serum levels of TG, TC and LDL-C of each dosage group were lower than the model group, while HDL-C is higher than the model group. Serum levels of TC, TG, LDL-C in high-dose berberine group and simvastatin group significantly decreased compared with model group (P<0.05). Serum lipid levels in high-dose berberine group were lower than medium-dose berberine group and low-dose berberine group, particularly of TG levels (P<0.05).3. Atherosclerosis plaque lesions in mice aortic morphological observation showed that there was no atherosclerosis lesion in control group. The atherosclerosis lesions of each dosage group were different degree of control compared with model group (P=0.00). The mice aorta atherosclerosis plaque area percentages of high-dose berberine group were significantly lower than medium-dose berberine group and low-dose berberine group (P<0.05). There was no significantly difference between low-dose berberine group and medium-dose berberine group (P>0.05). High-dose berberine group was not significantly different with simvastatin group (P>0.05).4. Visfatin expression in mice aorta atherosclerosis plaque:visfatin expressions in each dosage group were significantly higher than the model group (.P<0.05). High-dose berberine group was significantly lower in visfatin expression compared with low-dose berberine group and medium-dose berberine group (P<0.05). There was no significantly difference between high-dose berberine group and simvastatin group (P>0.05).5. Mice serum visfatin level:serum visfatin levels in simvastatin group, medium-dose berberine group, high-dose berberine group and low-dose berberine group significantly decreased compared with model group (P=0.00). Serum visfatin levels in low-dose berberine group were significantly higher than medium-dose berberine group and high-dose berberine group (P<0.05). There was no significantly difference between high-dose berberine group and simvastatin group (P>0.05). There was no significantly difference between high-dose berberine group and medium-dose berberine group (P=0.062).6. The expression of visfatin protein in mice aortic:The expressions of visfatin protein in each dosage group were lower than the model group (P<0.05). The expressions of visfatin protein in simvastatin group significantly decreased compared with low-dose berberine group (P=0.00). The expressions of visfatin protein in simvastatin group significantly increased compared with high-dose berberine group (P=0.00). There was no significant difference in in mice aortic visfatin protein between simvastatin group and medium-dose berberine group (P=0.166).7. The plasma levels of IL-6and TNF-a:the plasma levels of IL-6and TNF-a in each dosage group mice were significantly lower than the model group (P=0.00). There was no significant difference in plasma levels of IL-6among simvastatin group, high-dose berberine group, medium-dose berberine group and low-dose berberine group (P>0.05). There was no significant difference in plasma levels of IL-6between medium-dose berberine group and low-dose berberine group (P=0.277). There was no significant difference in plasma levels of IL-6between medium-dose berberine group and high-dose berberine group (P=0.343). There was no significant difference in plasma levels of TNF-a among simvastatin group, high-dose berberine group, medium-dose berberine group and low-dose berberine group (P>0.05). Plasma levels of TNF-a in simvastatin group were significantly lower than medium-dose berberine group and low-dose berberine group (P<0.05). There was no significant difference in plasma levels of TNF-a between medium-dose berberine group and low-dose berberine group (P>0.05).8. The influence of visfatin on HUVEC proliferation:visfatin concentration gradient group results show that compared with the control group,50,100,150,200μg·L-1visfatin can significantly inhibit HUVEC proliferation (P<0.05), there was no significant difference among100,150,200μg·L-1visfatin group (P>0.05). Visfatin time gradient group results show that compared with the control group,100μg·L-1visfatin stimulate for12h,24h and48h can significantly inhibit HUVEC proliferation (P<0.05).9. The interventive effect of berberine and inhibitors of MAPKs pathway on HUVEC proliferation induced by visfatin:compared with visfatin group,50and100μmol·L-1berberine can significantly reduce HUVEC proliferation inhibition induced by visfatin (P=0.00), and there was no significant difference between these two group (P>0.05). PD98059, SB203580and SP600125can significantly reduce the HUVEC proliferation inhibition induced by visfatin (P<0.05) and there was no significant difference among these three groups (P>0.05). PD98059, SB203580and SP600125were no significant difference compared with berberine (P>0.05).10. The influence of visfatin on p-ERK1/2expression in HUVEC:concentration gradient group results show that compared with the control group,50μg·L-1visfatin can significantly increased p-ERKl/2expression after24h stimulation (P=0.00),100and200μg·L-1visfatin p-ERKl/2expression in HUVEC higher than50μg·L-1visfatin groups have significant difference (P=0.00), and the amount to the concentration effect relationship; time gradient group results show that compared with the control group,100μg·L-1visfatin stimulate for24h and48h can significantly higher in HUVEC p-ERK1/2expression than12h group (P=0.00), and there was no significant difference between these two group (P=0.236).11. The influence of visfatin on p-p38MAPK expression in HUVEC: concentration gradient group results show that compared with the control group,50μg·L-1visfatin can significantly increased p-p38MAPK expression after24h stimulation (P=0.016), p-p38MAPK expression reach to peak after100μg·L-1visfatin stimulate for24h (P=0.00), there was no significant difference between100μg·L-1visfatin group and200μg·L-1visfatin (P=0.317). Time gradient group results show that compared with the control group,100μg·L-1visfatin stimulate for12h can significantly higher in HUVEC p-p38MAPK expression (P=0.003), p-p38MAPK expression reach to peak after100μg·L-1visfatin stimulate for24h (P=0.00).12. The influence of visfatin on p-JNK expression in HUVEC:concentration gradient group results show that compared with the control group,100and200μg·L-1visfatin can significantly increased p-JNK expression after24h stimulation (P=0.00), and there was no significant difference between these two group (P=0.593). Time gradient group results show that compared with the control group,100μg·L-1visfatin stimulate for12h can significantly higher in HUVEC p-JNK expression (P=0.001). P-JNK expression reach to peak after100μg·L-1visfatin stimulate for24h (P=0.00).13. TNF-a and IL-6secreted in HUVEC induced by visfatin:concentration gradient group results show that compared with the control group,100,200μg·L-1visfatin respectively stimulate for24h can significantly induce HUVEC secreted TNF-a and IL-6(P=0.00). Time gradient group results show that compared with the control group,100μg·L-1visfatin stimulate for6h can obviously induced HUVEC secreted TNF-a and IL-6(P=0.001). Induced HUVEC secreted TNF-a and IL-6reach to peak after100μg·L-1visfatin stimulate for24h (P=0.00). 14. The interventive effect of berberine and inhibitors of MAPKs pathway on p-ERKl/2、p-p38MAPK、JNK expression in HUVEC:compared with the visfatin group, berberine and PD98059can significantly reduce p-ERK1/2expression in HUVEC (P=0.00), berberine and SB203580can significantly reduce p-p38MAPK expression in HUVEC (P=0.00), berberine and SP600125can significantly reduce p-JNK expression in HUVEC (P=0.00).15. The interventive effect of berberine and inhibitors of MAPKs pathway on TNF-a and IL-6secreted in HUVEC induced by visfatin:compared with the control group,50μmol·L-1berberine beforehand processing HUVEC1h can obviously reduce the secretion of TNF-a and IL-6levels in HUVEC (P=0.00). PD98059, SB203580, SP600125can significantly reduce inflammatory cytokines TNF-a and IL-6secreted in HUVEC induced by visfatin (P<0.05).Conclusion:1. Berberine had the effects of lipid-lowering and weight-lossing for atherosclerosis mice.2. The expressions of visfatin were significantly high in atherosclerosis mice aortic and atherosclerosis plaques, berberine inhibited the formation of plaque lesions in ApoE-/-mice aorta, and decreased the expression of visfatin in atherosclerosis mice aortic and atherosclerosis plaque.3. The serum visfatin level was significantly high in atherosclerosis mice, berberine can decrease the serum visfatin level in atherosclerosis mice.4. The plasma levels of IL-6and TNF-a were significantly high in atherosclerosis mice, berberine can decrease the plasma levels of IL-6and TNF-a in atherosclerosis mice.5. Visfatin is concentration and time dependence on HUVEC proliferation and can induce HUVEC secreted IL-6and TNF-a, its mechanism may be through the activation of p38MAPK, ERK1/2and JNK pathway. Berberine can significantly reduce the inhibition of HUVEC proliferation induced by visfatin, berberine can obviously reduce the inflammatory cytokines IL-6and TNF-a secreted in HUVEC by visfatin, may be related to inhibition of p38MAPK, ERK1/2and JNK pathway.In conclusion, animal experiments confirmed that the atherosclerosis mice with hyperlipemia, high plasma levels of inflammation factors IL-6and TNF-a and high serum levels of visfatin, berberine can delay the development of atherosclerosis by reducing serum lipids, inhibiting the development of atherosclerosis plaque, reducing the contents of IL-6and TNF-a, down-regulating the expression of visfatin. Cell experiments determined that visfatin can injury endothelial cells and induce endothelial cells secrete inflammatory factor, berberine can delay the development of atherosclerosis by protecting endothelial cells and reducing the secretion of inflammatory factors, its mechanism might be related to inhibiting MAPKs pathways.