Effects Of Ruan Mai Ling On The Vascular Function In SHR And On The Cytoskeleton Proteins In VSMCs

Objective:1.To investigate effects of Ruaimailing(RML) on the vasomotion of thoracic aorta andmesenteric arteries in spontaneously hypertensive rats(SHR).2. To observe effects of Ruanmailing component of traditional Chinese medicine on thereorganization of cofilin, twinfilin-1, profilin-1induced by platelet-derived growthfactor(PDGF) in vascular smooth muscle cells(VSMCs)from rats.Method：Part Ⅰ32twelve-week-old male SHRs were divided into four groups randomly: untreatedcontrol(SHR,n=8), high-dose Ruanmailing (RH,3ml/Kg/d,n=8), low-dose Ruanmailing（RL,1ml/Kg/d,n=8）, losartan(LOS,10mg/Kg/d). Ruanmailin（gRML），a Chinese herbalcompound，together with losartan were given by gavage for12weeks separately, whileSHR control and8age-and sex-matched normotensive Wistar Kyotos rats (WKY)weregiven water only. Systolic blood pressure(SBP) was examined by tail cuff. Left ventriclemass to body weight ratio(LVM／BW) was calculated on sacrifice. Both the wall tolumen area ratios (W/L)and the thickness of the wall to the radius of the lumen(TW/R1)of and mesenteric arteries(3rdgarde branch)were assessed. Vascular reactivity to thevasoactive substance were researched by isolated rings of the thoracic aorta andmesenteric arteries. The potency of vascular contraction and relaxation was expressed asthe pD2value，where pD2is the negative logarithm of the agonist concentrationproducing50%of the maximum response.Part ⅡCultured VSMCs were derived from rat thoracic aorta. VSMCs were identified by-actin monoclonal antibody. Ruanmailing serum was prepared from rats after5days of drug garage. The rearrangement of cofilin, twinfilin-1and profilin-1wererespectively detected by Dylight488, Alexa Flour633and Cy3labeled antibodies underconfocal microscope.Results：Part Ⅰ1. After12weeks treatment with RML，SBP in both groups was markedly lower thanthat in SHR Group（P＜0.01）.2. LVM/BW in the RH group was significantly reduced than that in SHR group（P＜0.01), while the RL group was no statistical difference（P＞0.05）.3.W/L of mesenteric artery was significantly reduced in both the RL and RH group（P＜0.01). TW/R1was markedly decreased only in RH group（P＜0.01), while it was nostatistical difference in RL group（P＞0.05）.4.Acetyl choline(Ach)-induced endothelium-dependent Relaxation (EDdR) in throacic aortawas siginificantly decreased in SHR group compared with WKY, and it was markedlyimproved in RH and LOS groups (P＜0.01), while no statistical difference in RL group（P＞0.05）. Both sodium nitroprusside (SNP)-induced endothelium-independent relaxation(EDiR) and noradrenaline (NE)-induced vasoconstrictive function(VCF) was no statisticaldifference among all these groups.5. BothAch-induced EDdR and SNP-induced EDiR in mesentery arteries was siginificantlyreduced in SHR group in comparision with WKY, while it was markedly ameliorated in RHand LOS groups(P＜0.01), and no statistical difference was found in RL group. NE-inducedVCF was significantly inhanced in SHR group compared with WKY and it was markedlyattenuated in RH and LOS groups.Part Ⅱ1. Effects of Ruanmailing on the actin and cofilin in VSMCs.Compared with the control, after PDGF stimulation, VSMCs became fusiform. Theactin cytoskeleton was remodeled. The stress fibre were distributed intensively incytoplasm. The expression of lamellipodia became obvious. The expression of cofilinwas also increased. The co-localization of cofilin and actin was shown in cytoplasm and lamellipodia. They were most expressed at the leading edge of VSMC in fibrillar form.However, after treatment with10%Ruaimailing, the actin filament was depolomarized.The stress fibre was decreased greatly and arranged loosely. Both the lamellipodia andcofilin was lessened significantly. Much less actin filament and cofilin was distributedin the lamellipodia.2. Effects of Ruanmailing on reorganization of cofilin, twinfilin-1and profilin-1induced by PDGF in VSMCs.In comparison with the control, after the intervention by PDGF, VSMC became polygon.The expression of lamellipodia was enhanced visibly. The three actin binding proteinswere elevated and redistributed in cytoplasm. The range of cofilin and twinfilin-1was evenextended to the lamellipodia area at the leading edge of VSMC from the prior cytoplasmicregion near to the cell nucleus. Beyond the enhanced expression zone in cytoplasm, theexpression of profilin-1was also upregulated in the lamellipodia. Whereas, aftermanagement with10%Ruaimailing, the expression of all the three proteins were declinedsignificantly and discontinued,“spot”“sand”“honeycomb” accumulated in cytoplasm.Conclusion：1. Systolic blood pressure was markedly decreased with the RML treatment.2. RML could inhibit cardiovascular remodeling in SHR.3. RML could improve endothelium functions of thoracic aorta and mesenteric arteryin SHR. And it could also attenuate VCF of mesenteric artery in SHR.4. The expression of cofilin, twinfilin-1and profilin-1could be upregulated by PDGF.5. Except that the stress fibre and lamellipodia could be descreased by Ruanmailing, itwas also involved in regulating the process of polymerization and depolymerizationof the actin filament through the inhibition and redistribution of cofilin, twinfilin-1and profilin-1to suppress the morphological changes induced by PDGF in VSMC.