Role And Mechanism Of Actin Of KLF5 And Tongxinluo In Vascular Neointima Hyperplasia

Objective: Proliferation and migration of macrophages during neointima formation induced by arterial injury representa critical component of restenosis after angioplasty of human coronary arteries.Therefore,elucidation of novel cellular and molecular mechanisms that influence the pathogenesis of neointima formation could lead to development of new therapeutic approaches and insight into disease pathophysiology.effective inhibition of macrophage proliferation is of great significance for the prevention of atherosclerosis.Krüppel-like factor 5(KLF5)is a member of KLF family which have the structure of the zinc finger,and involved in cell proliferation,migration,apoptosis and tissue remodeling of many physiological and pathological processes in various diseases such as cardiovascular,cancer.Tongxinluo(TXL)is a kind of compound extract of traditional Chinese medicine.It has been widely used in applications in clinical treatment of cardiovascular and cerebrovascular diseases,due to its good vascular protective effect.The aim of this study was to investigate the inhibitory effect and its molecular mechanism of Tongxinluo on the proliferation and migration of macrophages induced by arterial injury,thus further providing a new theoretical and experimental basis for its clinical application.Methods: KLF5 wt and KLF5ly-/-(macrophage-specific KLF5 knockout)mice were randomly divided to control group,carotid artery ligation group and carotid artery ligation plus Tongxinluo group.Hematoxylin and eosin staining was used to evaluate the morphological changes.Mac-2,KLF5 and PCNA in the injured arterial tissues were determined by immunohistochemical and immunofluorescence staining.In vitro cultured macrophage RAW264.7 were stimulated with TNF-alpha to induce macrophage inflammation,after cells were pretreated ornot with Tongxinluo.The expression of KLF5 and PCNA in macrophages was detected by Western blot.The change of cell migration ability was observed by wound healing assay,and the cell proliferation was measured by MTS assay.The ubiquitination level of KLF5 as well as the signaling pathways that affect the interaction between FBXW7 and KLF5 were detected by immunoprecipitation.Results:1 TXL inhibits neointima hyperplasia induced by carotid ligation via reducing the proliferation and migration of macrophagesC57BL/6J mice were randomly divided to control group,carotid artery ligation group and carotid artery ligation plus Tongxinluo group.Hematoxylin and eosin staining showed that compared with the control group(Con),significant intimal hyperplasia occurred in ligation group(Ligated)and the I /M ratio was significantly increased.Conversly compared to the ligation group,Tongxinluo treatment obviously deseased intimal hyperplasia.The results showed that Tongxinluo can inhibit intimal hyperplasia induced by artery injury.The wound healing assay showed that cell migration rate of TNF-α group was higher than that of control group,while cell migration of Tongxinluo group decreased significantly compared with TNF-α group,suggesting that Tongxinluo can inhibit the migration of RAW264.7 cells induced by TNF-α.MTS results showed that TNF-α could significantly induce macrophage proliferation,however,its inducing effect on proliferation could be abrogated by Tongxinluo pretreatment.These results suggested that Tongxinluo could reverse proliferation of RAW264.7 cells induced by TNF-α.2 TXL inhibits macrophage proliferation through suppressing KLF5 expression induced by carotid ligation and TNF-αDouble immunofluorescence staining of Mac-2 and KLF5 displayed that Mac-2 and KLF5 positive cells of carotid artery ligation group were significantly increased,while those in ligation plus Tongxinluo group were less than the ligation group.These results suggested that Tongxinluo inhibitsthe migration and proliferation of macrophages through inhibiting the expression of KLF5 in inflammatory conditions.In vitro cultured RAW264.7 cells were preincubated with Tongxinluo and then treated with TNF-α.Western blot results showed that the increased expression of PCNA and KLF5 induced by TNF-α could be abolished by Tongxinluo treatment.3 TXL inhibits neointima hyperplasia by decreasing the expression of KLF5Hematoxylin and eosin staining showed that compared with the pAd transduced mice,the intima of pAd-KLF5 transduced mice was significantly thickened,intima-media ratio(I/M)increased obviously.Tongxinluo treatment could reverse the vascular intimal hyperplasia induced by carotid artery ligation.Immunofluorescence staining showed that compared with the pAd-transduced mice,positive cells of Mac-2 and PCNA staining increased significantly in pAd-KLF5 transduced mice,while Tongxinluo treatment reduced Mac-2 and PCNA positive cells.KLF5ly-/-mice treated with ligation showed that compared with the wild type(KLF5wt),the intima hyperplasia of KLF5ly-/-mice was significantly decreased.Immunofluorescence staining revealed that positive staining cells for Mac-2 and PCNA in KLF5ly-/-mice were less than that of the wild type mice.These results suggested that KLF5 can promote macrophage proliferation and migration,while Tongxinluo treatment inhibits the intimal hyperplasia by inhibiting the expression of KLF5 in macrophages.4 TXL inhibits KLF5 overexpression-induced proliferation and migration of macrophagesWestern blot results showed that overexpression of KLF5 obviously promoted the expression of PCNA and KLF5 in RAW264.7 cells,but their expression level was downregulated in the Tongxinluo group.MTS and wound healing assay indicated that proliferation and migration of macrophage transfected with p Ad-KLF5 were greatly increased compared with pAd transfected cells.Tongxinluo treatment markedly reduced the proliferation andmigration of macrophages induced by KLF5 overexpression.These results suggest that overexpression of KLF5 can exerts the inhibilory effect on the proliferation and migration by decreasing the expression of KLF5.5 TXL reduces the stability of KLF5 by regulating the ubiquitination and sumoylation of KLF5To clarify the molecular mechanism of the inhibition of KLF5 expression by Tongxinluo,we detected the ubiquitination of KLF5 by coimmunoprecipitation(CoIP).The results indicated that,compared with the control group,the TNF-α inhibited the ubiquitination of KLF5,whereas Tongxinluo increased significantly KLF5 ubiquitination,suggesting that Tongxinluo can accelerate the degradation of KLF5 by promoting the KLF5 ubiquitination.Western blot and co-immunoprecipitation results showed that Tongxinluo could promote the expression of ubiquitin-related enzyme FBXW7 and its interaction with KLF5.Further study found that sumoylation of KLF5 was greatly enhanced in TNF-α group,but was significantly reduced in Tongxinluo group.Tongxinluo could decrease sumoylation of KLF5 induced by TNF-α.The signal pathway-related proteins were detected by Western blotting,and the results showed that TNF-α could promote the expression of p-Akt,p-NF-kappa B,thus activating these signaling pathways,and leading to upregulation of KLF5 expression.When PI3K/Akt and NF kappa B signaling pathway was blocked by their specific inhibitors,sumoylation level of KLF5 was reduced.These results indicated that,Tongxinluo could promotes the sumoylation of KLF5,enhances the interaction of KLF5 with Fbxw7 and reduces the KLF5 stability through the activation of PI3K/Akt and NF kappa B signaling pathway.Conclusions:1 TXL inhibits neointima hyperplasia induced by carotid ligation via reducing the proliferation and migration of macrophages.2 TXL inhibits macrophage proliferation through suppressing KLF5 expression induced by carotid ligation and TNF-α.3 TXL inhibits neointima hyperplasia by decreasing the expression ofKLF5.4 TXL inhibits KLF5 overexpression-induced proliferation and migration of macrophages.5 TXL reduces the stability of KLF5 by regulating the ubiquitination and sumoylation of KLF5.