Study On The Effect Of AKT/FKHRL1 Signaling In CXCL16 Induced Cultured Smooth Muscle Cells Proliferation And The Protective Effect Of Apocynum Venetum L

CXCL16, a recently discovered important transmembrane chemokine, is expressed in human atherosclerotic lesion. The proliferation in vascular smooth muscle cell is the most important characteristic of atherosclerotic pathological change. There is a close relationship between inflammation and atherosclerosis. A lot of inflammatory factors regulated smooth muscle cell proliferation via correlated signal transduction pathway, and induced the development of atherosclerosis. AKT/FKHRL1 signal transduction pathway is important in regulate cell proliferation and apoptosis. CXCL16 could not only induce SMC proliferation, but also influence phospho-AKT and phospho-FKHRL1. AKT/FHHRL1 signal transduction pathway is perhaps one of the mechanism how CXCL16 leads atherosclerosis. As the important component of traditional Chinese medicine, Apocynum venetum L(AV) is discovered in inhibiting SMC proliferation induced by CXCL16, also influence phospho-AKT and phospho-FKHRL1 expressing. Maybe this is the important mechanism of Apocynum venetum L resist AS.Method:Smooth muscle cell were incubated and treated with 50ng/L CXCL16 and different concenteations (0.8,0.4,0.2mg/L) of AV for 24 hours and AKT inhibitor for 1h.Then we checked the proliferation with Cytometry and MTT, the expression of phospho-AKT and phospho-FKHRL1 with immunohistochemistry .Result:1. Compared with control group, SMC proliferation and of CXCL16 group significantly increased (P<0.05), proliferation apex time at 4 days.2. After Incubating SMC with CXCL16 and different concenteations (0.8, 0.4, 0.2mg/L) of AV for 24 hours and AKT inhibitor for 1h, AKT inhibitor and AV can significantly decrease proliferation (P<0.05). The effect of AV1 (0.8mg/L) group is less infirmly than AV2 andAV3 group.3. After Incubating SMC with 50ng/L CXCL16 for 24 hours, compared with control group, CXCL16 can significantly upregulate the expression of phospho-AKT and phospho-FKHRL1 with immunohistochemistry (P<0.05).4. After Incubating SMC with CXCL16 and different concenteations (0.8, 0.4, 0.2mg/L) of AV for 24 hours, compared with CXCL16 group, AV2 and AV3 can significantly decrease the expression of phospho-AKT and phospho-FKHRL1 with immuno-histochemistry (P<0.05), but we can not conclude if it changed in a concentration dependent manner. AV1 does not influence the expression of phospho-AKT and phospho-FKHRL1 (P>0.05).Discussion:There is a close relationship between inflammation and atherosclerosis. Atherosclerotic plaque is an inflammation pathological change and there are many inflammation factors during the process. CXCL16 is a recently discovered important chemokine which expressed in macrophage of human atherosclerotic lesion. It plays an important role in the development of atherosclerosis.Many inflammatory factors can promote SMC proliferation and induce formation of AS, such as PDGF secreted by platelet and macrophage, PDGF-like growth factor and TNF auto-secreted by SMC. Compared with control group, SMC proliferation and of CXCL16 group significantly increased (P<0.05) in our experiment, proliferation apex time at 4 days. This result is consistent with other investigations. To prevent the formation of AS, perhaps it is important to prevent SMC proliferation induced by CXCL16.CXCL16 exclusively binds CXCR6, and induce intracellar Ca²⁺ affluxion effectually. Intracellar [Ca²⁺] is the initiating agent of trigger signal transduction of cell proliferation, includes proliferation, apoptosis, gene expression and so on. Recently, investigation indicate that many components of total flavonoids can inhibit Ca²⁺ inflow, to resist free radical and inhibit NO formation. In this experiements the reason AV inhibit SMC proliferation induced by CXCL16 maybe relate with inhibit Ca²⁺ inflow.The risk factor of cardiovascular disease can injury the function of blood vessel endothelium, and activate intracellar signal conduction pathway,induce intranuclear gene expression, regulate SMC proliferation. FORKHEAD is the important target-gene located downstream of PI3-K-AKT/PKB signal conduction pathway. Activated AKT can phosphorylation of FOXO1(FKHR), FOXO3a(FKHRL1), FOXO4(AFX), and inhibit them nuclear interchange, degrade them transcription activity, and inhibit cell apoptosis. Our experiment result demonstrate CXCL16 can phosphorylation of AKT and FKHRL1, and via AKT/FKHRL1 signal conduction pathway to promote SMC proliferation. It is important for us to understand the formation of AS.AV is a sort of Chinese traditional medicine, it has many active effects. Recently much more investigation on AV have put up than before in heare disease. We want to study how AV cure AS deeply. Compared with CXCL16 group, AV can significantly decrease the expression of phosphorylation of AKT and FKHRL1, illustrate AV inhibit SMC proliferation perhaps via AKT/FKHRL1 signal conduction pathway. Nowadays, many people consider ox-LDL is an important risk factor on inducing of atherosclerosis. CXCL16 is originally identified as a scavenger receptor for phosphatidylserine and oxidized lipoprotein, participate the intracellar lipid accumulation in the progress of AS. It is still a question whether AV inhibit SMC proliferation relate with inhibit formation of ox-LDL mediated by CXCL16.SMC proliferation is important procedure to AS. CXCL16 is a recently discovered important chemokine which influence AS, it can induce SMC proliferation via AKT/FKHRL1 signal conduction pathway. As a a sort of Chinese traditional medicine, many investigations of AV concentrate on disease of cardiovascular system. Our experiments found AV can inhibit SMC proliferation induced by CXCL16, and to resist AS. It is important for us to investigate the mechanism of AS.Conclusion:CXCL16 can induce SMC proliferation. AV can inhibit this effect. In nomal, SMC can not express phospho-AKT and phospho-FKHRL1. CXCL16 can increase the expression of phospho-AKT and phospho-FKHRL1. AV can inhibit this effect, decrease the expression phospho-AKT and phospho-FKHRL1. Thus through upregulating proliferation, affecting the expression of phospho-AKT and phospho-FKHRL1, CXCL16 can advance the progress of AS via AKT/FKHRL1. AV can restrain these effects of CXCL16. Then AV can restrain the progress of AS.