Tongxinluo Abrogates The Downregulation Of KLF5and Tight Junction Proteins Induced By Angiotensin Ⅱ

Objective: Tight junctions (TJ) are the most apical type of cell–cellcontact in the lateral membrane between polarised cells, such as those existingin endothelia and epithelia. TJ shows a wide variability of tightness indifferent organs, ranging from almost complete tightening of the paracellularcleft for solutes. The tight junction constitutes the barrier both to the passageof ions and molecules through the paracellular pathway and to the movementof proteins and lipids between the apical and the basolateral domains of theplasma membraneAngiotensin II (Ang II), an endogenous peptide hormone, plays criticalrole in the pathophysiological modulation of cardiovascular functions. Ang IIis the principle effector of the renin-angiotensin system for maintaininghomeostasis in the cardiovascular system. Ang II is now known to be apleiotropic hormone that uses multiple signaling pathways to influent mostprocesses that contribute to the development and progression of cardiovasculardiseases, ranging from hypertrophy,endothelial dysfunction, cardiacremodeling, fibrosis, and inflammation to oxidative stress.Krüppel-like factor5(krüppel-like factor5, KLF5) is a zinc-fingercontaining transcription factor, its C-terminus contains three zinc fingerdomains, a DNA binding domain mainly, and its N terminal mainly providestranscription activation,as a basic transcription factor, KLF5is widelyexpressed in different tissues.Tongxinluo in capsule form is a compound prescription formulatedaccording to the meridian theory of traditional Chinese, which is extractedfrom a mixture of12medicinal constituents. It was regeistered in the StateFood and Drug Administration of China for the treatment of angina pectoris in1996. Recent studies have demonstrated that TXL has potential therapeuticvalue for a variety of diseases, such as improving endothelial function, lowing lipid, anti-inflammation, preventing atherosclerosis and promotingangiogenesis. However, the role and mechanism of TXL protective effect onendothelial cells is unknown, In the present study, we sought to elucidate themechanism whether and how TXL regulates endothelial cell tight junctionproteins.Methods: The expression of KLF5and tight junction proteins wasdetected by Western blotting and real-time PCR analysis. siRNA targetingKLF5was used to knock down endogenous KLF5expression.Immunohistochemical staining was used to detect the expression of KLF5andtight junction proteins.Results:1Angiotensin Ⅱ inhibits KLF5and tight junction protein expressionincluding VE-cadherin,claudin,occludin and beta-cateninTo examine whether angiotensin Ⅱ influences KLF5and tight junctionprotein expression，HCMEC was treated with angiotensin Ⅱ and thenwestern blot analysis was performed. We found that angiotensin Ⅱsignificantly induced KLF5and tight junction protein expression. In addition,real-time PCR was used to detect mRNAs for KLF5and tight junctionproteins and similar results were obtained.2Tongxinluo can dose-dependently obrogate the downregulation ofKLF5and tight junction proteins induced by angiotensin ⅡTo examine whether Tongxinluo influences KLF5and tight junctionprotein expression，HCMEC was incubated with Tongxinluo for24hours，and then HCMEC was treated with angiotensin Ⅱfor24hours.Western blotanalysis was performed to detect tight junction protein expression.We foundthat Tongxinluo can abrogate the downregulation of KLF5and tight junctionproteins induced by angiotensin Ⅱ, with a dose-dependent manner.3Over-expression of KLF5can promote the expression of tight junctionproteins including VE-cadherin,claudin,occludin and beta-cateninTo determine whether KLF5can regulate tight junction protein expression,HCMEC was transfected with adenovirus, and Western blot analysis was performed. Interestingly, we found that over-expression of KLF5can promotethe expression of tight junction proteins. Furthermore, Silencing of KLF5expression inhibits the expression of tight junction proteins. In addition,real-time PCR was used to detect mRNAs for KLF5and tight junctionproteins, and similar results were obtained.4Knockdown of KLF5inhibits the upregulation of tight junctionproteins induced by TXLTo clarify whether TXL-induced tight junction protein expression isKLF5-dependent, HCMEC was incubated with Tongxinluo for24h and thenwas transfected with KLF5-specific siRNA (siKLF5), and western blotanalysis was performed to detect tight junction protein expression. The resultsshow that knockdown of KLF5reduced the up-regulation of tight junctionprotein expression induced by TXL, indicating that Tongxinluo induces tightjunction protein expression in a KLF5-dependent manner.Conclusions:1Angiotensin Ⅱ inhibits KLF5and tight junction proteinexpression.2Tongxinluo can abrogate the downregulation of tight junctionproteins induced by angiotensin Ⅱ.3Overexpression of KLF5promotes the expression of tight junctionproteins. Silencing of KLF5expression inhibits the expression of tightjunction proteins.4Knockdown of KLF5expression inhibits the upregulation of tightjunction protein expression induced by TXL.