Alkaloids From Nelumbinis Plumula (AFNP) Ameliorate Aortic Remodeling Via RhoA/ROCK Pathway

Aims: The protective role of alkaloids from Nelumbinis Plumula(AFNP)on the aorta during hypertension is not yet fully understood.We hypothesied that AFNP exerts protective effects against angiotensin II(Ang II)-induced hypertension by mediating Rho A/ROCK pathway and phenotypic switching during hypertension.In the present study,we evaluated the effect of AFNP on Ang II-induced actin cytoskeleton reorganization and aorta remodeling,as well as the involvement of Rho A/Rho-associated coiled kinase(ROCK)pathway in protecting against hypertension.Methods: 1.Male SHR rats were treated with saline or AFNP and the blood pressure was measured weekly following 8 weeks.2.HE staining was used to observe the wall thickness and ratio of MT/LD and MA/LA.3.Western blotting was used to determine the protein expression of collagen I and ?-SMA in aorta.4.The isolated rat thoracic aortic rings were pre-contracted with norepinephrine or potassium chloride or angiotensin II followed by different concentrations of AFNP treatment and then observed the vasorelaxation effect of AFNP.5.The isolated rat aortic rings were pre-treated with Nitric oxide synthase(e NOS)inhibitor L-nitroarginine methyl ester(L-NAME)and cyclooxygenase inhibitor indomethacin(INDO)followed by different concentrations of AFNP and then observed the vasorelaxation effect of AFNP.6.The vasorelaxation effects of AFNP on endothelium-denuded and endothelium intact aortic rings were investigated after contracted with NE.7.The isolated rat aortic rings were pre-treated with or without K+ channel inhibitor(TEA,4-AP,Gli,or Ba Cl2)followed by different concentrations of AFNP treatment and then observed the vasorelaxation effect of AFNP after constriction with NE.8.The effects of AFNP on Ca Cl2-induced extracellular calcium influx after constriction with NE were investigated.9.MTT assay was used to assess the effect of AFNP on Ang II-induced VSMCs proliferation.10.Fluorescence microscopy was used to observe the effect of AFNP on Ang II-induced F-actin cytoskeleton remodeling of VSMCs.11.Western blot analysis was used to determine the effect of AFNP on Ang II-induced Rho A transposition,ROCK activation and phosphorylation MYPT1.Results: 1.AFNP treatment attenuated the increase of blood pressure in SHRs.2.AFNP treatment ameliorated the wall thickness and ratio of MT/LD and MA/LA of thoracic aorta.3.AFNP treatment decreased collagen I expression and increased ?-SMA expression in aorta.4.AFNP significantly induced the vasorelaxation of thoracic aortic rings precontracted with norepinephrine or potassium chloride or angiotensin II in a concentration-dependent manner.5.AFNP treatment increased the relaxation of isolated thoracic aortic rings precontracted with NE,which was not attenuated by treatment with NG-nitro-L-argininemethylester(L-NAME)and indomethacin(INDO).6.AFNP induced the vasorelaxation of the endothelium-intact arteries and endothelium-denuded arteries,and the vasorelaxation effect of AFNP was endothelial independent.7.AFNP treatment increased the relaxation of isolated thoracic aortic rings precontracted with NE in an endothelium-independent manner,which was attenuated by treatment with 4-aminopyridine(4-AP)and glibenclamide(Gli),but not by treatment with Tetrathylamonium(TEA)or Ba Cl2.8.AFNP pretreatment attenuated the Ca Cl2-induced constriction of isolated thoracic aortic rings in Ca2+-free solutions after constriction with NE.9.AFNP treatment inhibited the proliferation of Ang II induced VSMCs in a concentration-dependent manner.10.AFNP treatment ameliorated Ang II induced F-actin cytoskeleton remodeling in VSMCs.11.AFNP treatment inhibited Ang II induced Rho A activation and decreased the protein expression of ROCK and the phosphorylation of MYPT1.Conclusion:AFNP can attenuate the elevation of blood pressure in SHRs.The vasorelaxation effects of AFNP on aorta was endothelial independent,which is likely mediated by inhibition of K+channel and extracellular calcium influx.And AFNP can ameliorate vascular remodeling by inhibiting cytoskeleton remodeling of VSMC and regulating Rho A/ROCK pathway.