Rab5a Mediated AT₁R Endocytosis Underlines Liensinine Mechanism Of Vascular Function Maintenance And Hypertension Therapy

ObjectiveThe project is aimed to explore the pharmacodynamic effects of Liensinine on counter hypertension and protecting normal vascular functions,and further investigate Liensinine effects on Ca²⁺,NF-κB,MAPK,and also TGF-β/Smads signaling,as along with Liensinine regulation on AT₁R intracellular transport.Rab5a mediated AT₁R endocytosis is a novel mechanism that Liensinine ameliorate hypertension and protect normal vascular function.We try to provide fundamental data that will set up theoretical basis for the clinical application of Liensinine on hypertension prevention and treatment,and provide ideas on new therapeutic targets of hypertension.Methods1.Antihypertensive and vascular protective effects of Liensinine:C57BL/6 hypertension model was established by AngⅡstimulation using subcutaneous osmotic pump.The groups were Control group,AngⅡgroup,AngⅡ+valsartan Val（10.4 mg/kg）,AngⅡ+Lien（2.5mg/kg）,AngⅡ+Lien（5 mg/kg）and AngⅡ+Lien（10 mg/kg）,with 12 mice in each group.Non-invasive blood pressure monitor,ultrasound machine,and HE staining were used to measure blood pressure,pulse wave propagation velocity and morphological changes of abdominal aorta in mice.The effect of liensinine on AngⅡ-induced increase of abdominal aortic vascular tone was measured by vascular tone detection system.ELISA and immunohistochemistry were used to identify the effect of Liensinine on inflammatory response in systemic circulation and vascular tissues in hypertension mice model.The effect of liensinine on hypertension induced vascular remodeling was examined by Sirius red staining and immunohistochemistry.2.The mechanism of Liensinine in protecting vascular function:Western Blot and immunohistochemistry were used to investigate the effect of AngⅡon vascular contraction,vascular inflammation and vascular remodeling through Ca²⁺signal,NF-κB signal,MAPK signal,TGF-β/Smads signal in hypertension model in vivo and vascular smooth muscle cell line in vitro.We further investigated the role of liensinine on AngⅡinduced signaling.We also examined the combination of endocytic inhibitor and liensinine’effect on vascular tone.3.The role of AT₁R endocytosis key molecule Rab5a in regulating hypertension:Rab5a knockout（KO）mice were constructed by Embryonic Stem Cell targeting and Rab5a overexpress（OV）mice were constructed by tail vein injection of adeno-associated virus.AngⅡwas used to induce hypertension model in Rab5a KO or Rab5a OV mice.Non-invasive blood pressure monitor,ultrasound machine were applied to monitor the blood pressure,the velocity of abdominal aortic pulse wave and HE staining was applied to observe morphological changes of hypertensive mice tissue.Rab5a knockdown cells were constructed by small interfering RNA,and the effect of si Rab5a on AT₁R endocytosis and intracellular transportation was further evaluated by Western Blot and confocal microscopy.4.Regulation of Liensinine on Rab5a expression and AT₁R intracellular transportation:q PCR was used to examine Rab5a m RNA transcription in vascular smooth muscle cells after liensinine intervention.Western Blot was used to examinethe Rab5a protein synthesis after treatment of liensinine along with synthase inhibitor or lysosomal degradation inhibitor.Confocal microscopy was used to observe AT₁R endocytosis.upon AngⅡsimulation either on combination with liensinineResult1.Antihypertensive and vascular function protective role of Liensinine:Liensinine ameliorates AngⅡinduced blood pressure increases in mice and significantly protect vascular functions including vasoconstriction,vascular inflammation,phenotypic transformation and vascular remodeling.2.The protective role of Liensinine on vascular function:In vivo and in vitro experiments showed that Liensinine could inhibit AngⅡinduced activation of Ca²⁺,NF-κB,MAPK and TGF-β/Smads signaling（P<0.05）.Liensinine also inhibited AngⅡinduced down-regulation ofα-SMA protein and up-regulation of PCNA protein（P<0.05）.Inhibition of clathrin mediated endocytosis is able to abrogate liensinine effect against AngⅡ-induced vascular tone increase.3.The mechanism of Rab5a,a key molecule of AT₁R endocytosis,in regulating hypertension:Rab5a deletion can further uplift AngⅡ-induced blood pressure increase,and exacerbate vascular function and pathological damage（P<0.05）,The possible molecular mechanism is through inhibition of AT₁R internalization and degradation;Consistently,Rab5a overexpression can inhibit AngⅡ-induced blood pressure increase and suppress the occurrence and development of vascular remodeling（P<0.05）.4.The regulatory effect of Liensinine on Rab5a expression and AT₁R intracellular transportation:Liensinine alone is able to increase Rab5a gene expression and protein synthesis in vascular smooth muscle cells;Thus Liensinine could promoted AngⅡ-induced AT₁R endocytosis in a time dependent manner.Conclusion1.Liensinine can not only inhibit the increase of blood pressure,but also improve the abnormal vascular function by suppressing vascular contraction,vascular inflammation,phenotypic transformation and vascular remodeling.2.The protective effect of Liensinine on vascular function may be through the regulation of Ca2+,NF-κB,MAPK and TGF-β/Smads signaling.3.Knockdown and overexpression of Rab5a revealed that Rab5a is a critical target for regulating AT1R intracellular transport and thereby regulating blood pressure.4.The signaling pathways mentioned abobe aredownstream of AngⅡ/AT1R signaling.Liensinine is able to promote AT1R endocytosis and inhibit the activation of AngⅡsignaling by increasing the gene transcription and protein expression of Rab5a.This investigation elucidated the Liensinine effect and its mechanism on anti-hypertension and the protection of normal vascular function.It was demonstrated for the first time that Liensinine could promote the intracellular transport of AT₁R by increasing the gene transcription and protein expression of Rab5a,which is a novel target and newly demonstrated mechanism of liensinine in anti-hypertension and vascular function protection.The regulatory effect of Rab5a on hypertension was demonstrated for the first time in both knockout and overexpression of hypertension mice.The results of this study can further enrich the knowledge of hypertension pathogenesis,bring up a new vision on the antihypertensive mechanism of liensinine,also,they provide more ideas and directions for the clinical treatment of hypertension,and provide experimental basis for the clinical application of Liensinine against hypertension.