The Mechanism Research Of Honokiol Inhibiting Carotid Artery Atherosclerotic Plaque Formation In Apolipoprotein E-knockout Mice

Objective:Atherosclerosis is a chronic inflammatory disorder involving the large and medium-sized arterial wall,which is characterized with lipid accumulation,monocytes or macrophages infiltration,vascular smooth muscle cells migration,and collagenous fibers hyperplasia.The mechanism of atherogenesis has been disputed for decades,with an agreement of several potential theories including oxidative stress,endothelial dysfunction,inflammatory effect,and immunological function disorder.Oxidative stress is considered to be involved in all the pathological processes of AS.Reactive oxygen species(ROS)production,resulted by oxidative and anti-oxidative unbalance,may cause the endothelial cell damage and the monocytes activation into macrophage.Moreover,excessive generation of ROS can induce the migration and proliferation of vascular smooth muscle cells,which aggravates intimal hyperplasia.All of these effects of ROS promote the progression of AS.NO,as an important bioactive molecule,is widely existed in various cells and tissues and considered to be the marker of endothelial dysfunction.Under physiological condition,NO functions in various aspects including,mediating vascular smooth muscle relaxation,dampening platelet aggregation,and reducing pulmonary circulation resistance,etc.However,in pathologic conditions,the excessive production of NO may cause tissue and organ damage.During the progression of AS,a lot of NO is produced by iNOS,which plays a key role in this process.Inflammation plays important roles in the occurrence and development of AS,which is related to a variety of cytokines,such as TNF-?,IL-6,and IL-1?.TNF-? is a pro-inflammatory cytokine and expressed in atherosclerotic lesions.IL-6 is a representative inflammatory cytokine and the level of IL-6 is significantly upregulated in local plaques of AS.IL-1? has also been proved to play important roles in AS.Toll-like receptors(TLRs),as “sensors” of initial immune response,are key players in the pathogenesis of inflammatory conditions including the process of atherogenesis.They are expressed by a variety of immune cells where they recognize pathogen-associated molecular patterns(PAMPs).TLRs recruit adaptor molecules to mediate activation of NF-?B pathways and associate with the development of atherosclerosis through various mechanisms.TLR4 is expressed in lipid-rich and atherosclerotic plaques.In TLR2-/-and TLR4-/-mice,atherosclerosis-associated inflammation was diminished.NF-?B is a transcription factor that regulates various inflammation-related genes expression.NF-?B binds to its inhibitory protein,I?B? and exists in cytoplasm almost inactively under normal conditions.In response to various stimuli,NF-?B translocates into the nucleus and binds to the promoters of its target genes to facilitate their expression.Previous studies showed that NF-?B played important roles in the regulation of inflammatory mediator expression during AS.Statins are selective competitive inhibitors of hydroxymethyl glutaric acid monoacyl coenzyme A(HMG-Co A)reductase.Their effects on regulating blood lipids and preventing AS-related cardiovascular and cerebrovascular accidents have been confirmed by a large number of studies.However,with the widespread use of statins,many clinical side effects related to statins have been found,such as rhabdomyolysis,new onset diabetes mellitus and hemorrhagic stroke.Although this does not affect its value in the prevention of cardiogenic and cerebral apoplexy,it is still of practical significance to further study the mechanism of the occurrence and development of AS and to develop new drugs to supplement existing clinical drugs for AS control.Honokiol is a natural active compound,extracted from the Chinese herbal medicine Magnolia officinalis,which has various pharmacological activities,such as anti-inflammatory,anti-thrombotic,and antitumor activities.A series of recent studies also show that honokiol may have an anti-atherogenic potentiality.Therefore,in this study,we constructed apolipoprotein E gene knockout(ApoE-/-)mouse carotid artery AS model to compare the effects of HNK and atorvastatin on the formation and development of AS in vivo for the first time,and confirmed firstly that HNK can inhibit the formation and development of AS.Methods:30 six weeks old Male ApoE-/-mice were divided into 5 groups,among which 6 in one group were fed with normal diet(ND)and 24 in 4 groups with western-type diet(WD)for ten weeks.The perivascular collar was surgically placed on the right common carotid arteries of the mice after two weeks of WD feeding,to accelerate the progression of atherosclerotic lesion.After that,the mice with WD were randomly divided into four groups and respectively treated with intraperitoneal injection of vehicle(200 ?l corn oil),10 mg/kg honokiol,20 mg/kg honokiol,and oral gavage of 10 mg/kg atorvastatin(ATV)once a day for eight weeks.The mice with ND or ATV were intraperitoneally injected with vehicle(200 ?l corn oil)in the same way.Eight weeks after surgery,the right common carotid arterie was excised,one part of which was fixed in 10% formaldehyde,and another of which was quickly frozen in liquid nitrogen for further tests.To determine the effect of honokiol on the progression of atherosclerotic plaque,we inspected the fixed section of carotid arteries with HE staining,Masson trichrome staining,and immunohistochemical staining of ?-SMA,detecting the degree of atheroma formation,collagen fiber hyperplasia,and vascular smooth muscle cells migration,respectively.We further investigated the effects of honokiol on ROS production,SOD activity and NO synthesis in carotid tissue,using commercial Reactive oxygen species(ROS)Assay Kit,Superoxide Dismutase(SOD)Assay Kit and NO Assay kit,respectively.The m RNA expressions in carotid tissue were detected by real-time PCR,and we researched whether the expression of TNF-?,IL-6,IL-1? and iNOS would be impacted by honokiol.Furthermore,Western blot analysis was used to deliberate the expressions of iNOS,TLR2,TLR4 and the components of canonical NF-?B signaling pathway.Moreover,EMSA assay was performed to assess the DNA-binding activity of NF-?B using NF-?B EMSA kit.All statistical analyses were performed by one-way analysis of variance(ANOVA)followed by Newman-Keuls' Multiple Comparison Test by Graph Pad Prism 5 software.A statistically significant difference was found when the p value was less than 0.05.Results:In carotid artery tissues,obtained from apo E-/-mice feeding by western-type diet and placing with perivascular collar around the right carotid artery,we observed a series of significant activations of oxidative stress,vascular endothelial dysfunction,inflammatory reaction,initial immune response and NF-?B signaling pathway,which could be demonstrated by the increasing of ROS synthesis and SOD consumption,the over-expressing of iNOS and excessive NO synthesized by which,the transductions of inflammatory cytokines(TNF-a,IL-6,IL-1?),the over-expressions of TLRs(TLR2 and TLR4),the enhancing DNA binding activity of NF-?B,the raised amount of p-I?B? and nuclear NF-?B p65,as well as the reduced levels of I?B? and cytoplasm NF-?B p65.All above were associated with the pathological changes following the progression of atherosclerosis,including lipid deposition,macrophage/foam cell accumulation,collagen hyperplasia,and proliferation or migration of the differentiated VSMCs.In our research,we found that HNK and ATV could alleviate the cascade above with the details as follow.1.HNK postponed the progression of AS plaque in the carotid artery of ApoE-/-mice.(1)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV reduced the plaque load significantly(p < 0.05,P < 0.0001 and P < 0.0001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.0001),while the effect of 20mg/kg HNK was similar to that of 10mg/kg ATV(p > 0.05).(2)20 mg/kg HNK and 10 mg/kg ATV inhibited the synthesis of collagen fibers by vascular smooth muscle cells significantly(p < 0.001 and P < 0.0001,respectively).Among them,10 mg/kg ATV had the same effect as 20 mg/kg HNK(p > 0.05),but stronger than 10 mg/kg HNK(p < 0.05).(3)20 mg/kg HNK and 10 mg/kg ATV inhibited the differentiation of vascular smooth muscle cells significantly(p < 0.05).Among them,20 mg/kg HNK had the same effect as 10 mg/kg ATV(p > 0.05),and both of which seemed to be stronger than 10mg/kg HNK,but there was no significant difference between them.2.HNK restrained the oxidative stress in atherosclerotic artery.(1)20 mg/kg HNK and 10 mg/kg ATV reduced the content of ROS significantly(p< 0.05),even reach the level of normal diet group(p > 0.05).Among them,20 mg/kg HNK had the same effect as 10 mg/kg ATV(p > 0.05),and seemed to be stronger than10 mg/kg HNK,but there was no significant difference between them.(2)20 mg/kg HNK and 10 mg/kg ATV reduced the consumption of SOD significantly(p < 0.001 and P < 0.05,respectively).Among them,the effect of HNK was dose-dependent(p < 0.05),while the effect of 10mg/kg ATV was similar to that of10mg/kg or 20mg/kg HNK(p > 0.05).3.HNK alleviated the endothelial dysfunction in atherosclerotic artery by downregulating the over-expression of iNOS and reducing the synthesis of NO.(1)20 mg/kg HNK and 10 mg/kg ATV reduced NO synthesis significantly(p <0.001),even reach the level of normal diet group(p > 0.05).Among them,the effect of HNK was dose-dependent(p < 0.05),while the effect of 10mg/kg ATV was similar to that of 20mg/kg HNK(p > 0.05),but stronger than that of 10mg/kg HNK(p < 0.001).(2)10mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited iNOS gene transcription significantly(p < 0.0001,P < 0.0001 and P < 0.001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.05),while the effect of 10mg/kg ATV was similar to that of 10mg/kg or 20mg/kg HNK(p > 0.05).(3)10mg/kg,20mg/kg HNK and 10mg/kg ATV inhibited iNOS protein synthesis significantly(p < 0.0001).Among them,the effect of HNK was dose-dependent(p <0.0001),while the effect of 10mg/kg ATV was stronger than that of 10mg/kg or 20mg/kg HNK(both P < 0.0001).(4)10mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited the expression and abnormal distribution of iNOS protein in tissues significantly(p values were p < 0.05,P< 0.001 and P < 0.001 respectively),and the latter two groups were even close to the normal diet group(p values were > 0.05).Among them,20 mg/kg HNK had the same effect as 10 mg/kg ATV(p > 0.05),and seemed to be stronger than 10 mg/kg HNK,but there was no significant difference between them.4.HNK eliminated vascular inflammation by inhibiting the expression of inflammatory cytokines in atherosclerotic artery.(1)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited the transcription of TNF-? significantly(p < 0.05,P < 0.0001 and P < 0.05,respectively).Among them,the effect of HNK was dose-dependent(p < 0.001),while the effect of 10mg/kg ATV was similar to that of 10mg/kg HNK(p > 0.05),but significantly weaker than that of 20mg/kg HNK(p < 0.001).(2)20mg/kg HNK and 10 mg/kg ATV inhibited the transcriptional level of IL-6significantly(p < 0.0001 and P < 0.001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.001),while the effect of 10mg/kg ATV was similar to that of10mg/kg HNK(p > 0.05),but weaker than that of 20mg/kg HNK(p < 0.05).(3)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited the level of IL-1?transcription significantly(p < 0.05,P < 0.0001 and P < 0.001,respectively).Although the effect of 20mg/kg HNK seemed to be stronger than that of 10mg/kg ATV and10mg/kg HNK,there was no significant difference among the three groups.5.HNK inhibited the expression of TLR2 and TLR4 in atherosclerotic artery..(1)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited the expression of TLR2significantly(p < 0.001,P < 0.0001 and P < 0.0001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.001),while the effect of 10mg/kg ATV was stronger than that of 10mg/kg HNK(p < 0.0001)and 20mg/kg HNK(p < 0.05).(2)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV inhibited the expression of TLR4significantly(p < 0.001,P < 0.0001 and P < 0.0001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.001),while the effect of 10mg/kg ATV was stronger than that of 10mg/kg HNK(p < 0.0001)and 20mg/kg HNK(p < 0.05).6.HNK repressed the activation of NF-?B signaling pathway in atherosclerotic arte-ry.(1)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV reduced the level of p-I?B?protein(p < 0.0001),and increase the level of I?B? protein significantly(p < 0.001,P <0.0001 and P < 0.0001,respectively).Among them,the effect of HNK was dose-dependent(p < 0.001),while the effect of 10mg/kg ATV was stronger than that of10mg/kg HNK(p < 0.0001)and 20mg/kg HNK(p-I?B?: P > 0.05;I?B?: P < 0.0001).(2)10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV increased the protein level of NF-?B p65 in cytoplasm(p < 0.001,P < 0.0001 and P < 0.0001 respectively),but decrease the protein level of NF-?B p65 in nucleus significantly(p < 0.0001).Among them,the effect of HNK was dose-dependent(p < 0.0001),while the effect of 10mg/kg ATV was stronger than that of 10mg/kg HNK(p < 0.0001)and 20mg/kg HNK(p <0.0001).(3)For the sake of sample size,only one group of DNA binding activity of NF-?B was detected.The results showed that the DNA binding activity of NF-?B decreased in the order of 10 mg/kg,20 mg/kg HNK and 10 mg/kg ATV.Conclusion:The results of this study proved once again that western-type diets and carotid cannula can induce oxidative stress,endothelial dysfunction,inflammation reaction,immune response and activation of NF-?B signaling pathway system in carotid artery wall of ApoE-/-mice,thereby promote the occurrence and development of AS lesions.This study demonstrated firstly in vivo that HNK and ATV can slow down the development of AS by eliminating oxidative stress,improving vascular endothelial dysfunction,down-regulating the expressions of inflammatory cytokines,limiting the activation of the initial immune system and inhibiting the NF-?B signaling pathway.The effect of HNK on TLR was discussed for the first time,suggesting that HNK has pharmacological activity in immunoregulation.In addition,this study found that the above pharmacological effects of HNK were dose-dependent.Meanwhile,for the first time,this study found that 20mg/kg HNK was weaker than10mg/kg ATV in inhibiting the activation of TLRs and NF-?B signaling pathway system,but stronger in down-regulating the expression of inflammatory cytokines.In terms of anti-oxidative stress,improving endothelial function and other pharmacological effects,the two effects are similar.All above would demonstrate that HNK has multiple antiatherogeic effects,and needs further investigation in human body as a reasonable medicine choice for prohibiting the progression of atherosclerotic diseases.