Effects Of Polygonum Multiflorum Thunb And Its Bioactive Ingredient Tetrahydroxystilbene Glycoside On Microvascular Endothelial Dysfunction And Arterial Blood Pressure In Obesity-associated And Spontaneously Hypertensive Rats And Underlying Mechanisms

BackgroundCardiovascular diseases are the most serious threat to public health with high mortality rate and arterial hypertension is an independent risk factor for cardiovascular diseases.Endothelial dysfunction contributes to increased peripheral vascular resistance and subsequent hypertension.Furthermore,obesity is a major risk for hypertension.Obesity impairs vasodilating properties of the vascular endothelium,which promotes changes in the patterns of the flow and pressure of blood,thereby leading to endothelial dysfunction and obesity-related hypertension.Autophagy,an evolutionarily conserved catabolic process,refers to the delivery and degradation of cytoplasmic material,which is considered a cellular recycling mechanism.Autophagy regulates endothelial function,however,whether autophagy is related to endothelial dysfunction and hypertension in obesity remains largely unclear.2,3,5,4'-tetrahydroxystilbene-2-O-?-D-glycoside(TSG)is the main bioactive ingredient extracted from the traditional Chinese medicinal herb Polygonum multiflorum Thunb.which has been used safely in China for approximately two millennia,and has been reported to have a wide spectrum of pharmacologic functions.TSG has been reported to help people live longer,protect impaired neural networks,improve blood flow,prevent the production of oxygen radicals and protect against oxidized LDL-induced endothelial dysfunction.Nonetheless,little is known about the effects of TSG on endothelial dysfunction in obesity.ObjectivesWe wished to ascertain:(i)the role of autophagy in endothelial dysfunction in obesity-associated and spontaneously hypertensive rats and the underlying mechanisms;(ii)whether Polygonum multiflorum Thunb.and its bioactive ingredient tetrahydroxystilbene glycoside(TSG)influences endothelial dysfunction as well as arterial blood pressure in obesity-associated and spontaneously hypertensive rats.Methodsa)BP measurement: Systolic blood pressure(SBP)was measured non-invasively with a tail-cuff system in accordance with standard procedures.Rats and mice were allowed to acclimatize on a heating mantle at 39°C for ?10 min before measurement.b)Intraperitoneal glucose tolerance test(IPGTT): After a 16 h fast,alert rats were challenged with a glucose load of 2 g/kg body weight administered I.P.for the glucose tolerance test.Tail blood was taken 0,30,60,90,120 min after administration of the glucose load,and glucose measurements were determined by using an ACCU-CHEK optium blood glucose meters(Roche,China).c)Experiments undertaken in organ chambers: Animals were anesthetized and mesenteric arterioles of rats were dissected immediately after killing.To weed out those devitalized artery segments,the rings were contracted with 60 mmol/L KCl solution before the use of phenylephrine.Once a stable contraction was achieved,an endothelium-dependent vasodilator,ACh was added to the perfusate to determine endothelial function.The procedure was repeated with the endothelium-independent vasodilator SNP to determine the function of vascular smooth muscle.Relaxation in response to each drug was indicated as the percentage of phenylephrine contraction,and 100% relaxation denoted when th active tone returned to the baseline level.d)Cell culture and treatments: Experiments using HUVECs were carried out at passages 3–8.HUVECs were exposed to palmitate(500 ?M)or bovine serum albumin for 24 h.Then,cells were treated with 100 ?M TSG,autophagy-inducer rapamycin(100 n M)or IGF-1.e)Transfection with the adenovirus-red fluorescent protein–green fluorescent protein–microtubuleassociate protein 1 light chain 3(Ad-m RFP–GFP–LC3)expression vector and Akt/m TOR knockdown: HUVECs were transfected with the Ad-m GFP–RFP–LC3 adenovirus(multiplicity of infection = 50)and,36 h later,the cells were imaged under a confocal microscope.In addition,cells were transfected with mechanistic target of rapamycin(m TOR)or Akt small interfering RNA by Lipofectamine? RNAi MAX.The efficiency of gene knockdown was detected using western blotting 48 h after transfection.f)Western blotting: Proteins were separated on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes.Membranes were blocked with bovine serum albumin and incubated with antibodies.Antibody binding was detected via enhanced chemiluminescence,and membranes were scanned with Chemi Doc XRS.g)Statistical analysis: Data are the mean ± SEM and were analyzed by a two-tailed Student's t-test between two groups or by one-way ANOVA if three or more groups were compared.In all statistical comparisons,P < 0.05 was considered significant.All statistical tests were undertaken with Graph Pad Prism v6.0.Resultsa)Systolic blood pressure(SBP)of obese ZDF rats was significantly higher than in control lean ZDF rats.Polygonum multiflorum Thunb.extract(PME,250 mg/kg/day)and TSG(100 mg/kg/day)administration could significantly postpone the process of hypertension.No significant changes in arterial SBP were observed after intervention with emodin(80 mg/kg/day).Vascular function results showed that the endothelium-dependent vasodilatation function of mesenteric arterioles was significantly decreased in ZDF rats,and PME and TSG treatment could significantly improve the microvascular endothelium relaxation function of ZDF rats in different degrees,and the effect of TSG was more significant,while emodin was not.It is suggested that TSG may play a major role in the microvascular protection process of ZDF rats.b)Compared with their lean counterparts,obese ZDF rats exhibited hypertension and endothelial dysfunction,along with impaired Akt/m TOR signaling and upregulated expression of autophagy-associated proteins beclin1,microtubule-associated protein 1 light chain 3 II/I,autophagy protein(Atg)5 and Atg 7.Two-week TSG(100 mg/kg/day)administration restored blood pressure and endothelial function,reactivated Akt/m TOR pathway and decreased endothelial autophagy in ZDF rats.Rapamycin pretreatment blocked the hypotensive effect of TSG in ZDF rats.Suppression of Akt/m TOR expression with si RNA significantly blunted the anti-autophagic effect of TSG(100 ?mol/L)in HUVECs as evidenced by abnormal autophagic flux and increased expression of autophagy-associated proteins.c)Compared with Wistar Kyoto rats(WKY),young and adult SHRs showed endothelial dysfunction of the aorta and mesenteric artery,along with decreased p Akt,pm TOR,and autophagic marker protein p62 and increased LC3 II/I in microvascular but not aortic tissues.TSG(100 mg/kg/day)administration for 14 days significantly improved mesenteric vascular endothelial function,increased levels of p Akt and pm TOR,and decreased autophagy.Pretreatment of young SHRs with the m TOR inhibitor rapamycin blocked the antiautophagic and vasodilative effects of TSG.Moreover,TSG(100 ?mol/L)significantly activated Akt-m TOR signaling in HUVECs and reduced the autophagic levels in vitro,which were almost completely blocked by rapamycin.ConclusionsIn this study,we found that 1)PME significantly improves microvascular endothelial dysfunction in obese ZDF rats and effectively reduces their arterial blood pressure.After further screening its active ingredients,TSG plays a key role on PME's hypotensive effects.2)excessive autophagy is one of the mechanisms underlying the microvascular endothelial dysfunction in obese-associated and spontaneous hypertensive rats.TSG treatment improves microvascular endothelial dysfunction via an autophagy pathway and ameliorates obesity-increased arterial BP rather than spontaneous hypertension,suggesting the possible effects of TSG on subclinical lesion of cardiovascular events and that could be an anti-hypertension drug candidate in obesity-associated cardiovascular diseases.